RNA interference mediated inhibition of stearoyl-CoA desaturase (SCD) gene expression using short interfering nucleic acid (siNA)

ABSTRACT

This invention relates to compounds, compositions, and methods useful for modulating Stearoyl-CoA desaturase (SCD) gene expression using short interfering nucleic acid (siNA) molecules. This invention also relates to compounds, compositions, and methods useful for modulating the expression and activity of other genes involved in pathways of Stearoyl-CoA desaturase (SCD) gene expression and/or activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of Stearoyl-CoA desaturase (SCD) genes.

This application is a continuation-in-part of International PatentApplication No. PCT/US03/04317, filed Feb. 13, 2003, which claims thebenefit of U.S. Provisional Application No. 60/412,304, filed Sep. 20,2002. This application is also a continuation-in-part of InternationalPatent Application No. PCT/US04/16390, filed May 24, 2004, which is acontinuation-in-part of U.S. patent application Ser. No. 10/826,966,filed Apr. 16, 2004, which is continuation-in-part of U.S. patentapplication Ser. No. 10/757,803, filed Jan. 14, 2004, which is acontinuation-in-part of U.S. patent application Ser. No. 10/720,448,filed Nov. 24, 2003, which is a continuation-in-part of U.S. patentapplication Ser. No. 10/693,059, filed Oct. 23, 2003, which is acontinuation-in-part of U.S. patent application Ser. No. 10/444,853,filed May 23, 2003, which is a continuation-in-part of InternationalPatent Application No. PCT/US03/05346, filed Feb. 20, 2003, and acontinuation-in-part of International Patent Application No.PCT/US03/05028, filed Feb. 20, 2003, both of which claim the benefit ofU.S. Provisional Application No. 60/358,580 filed Feb. 20, 2002, U.S.Provisional Application No. 60/363,124 filed Mar. 11, 2002, U.S.Provisional Application No. 60/386,782 filed Jun. 6, 2002, U.S.Provisional Application No. 60/406,784 filed Aug. 29, 2002, U.S.Provisional Application No. 60/408,378 filed Sep. 5, 2002, U.S.Provisional Application No. 60/409,293 filed Sep. 9, 2002, and U.S.Provisional Application No. 60/440,129 filed Jan. 15, 2003. Thisapplication is also a continuation-in-part of International PatentApplication No. PCT/US04/13456, filed Apr. 30, 2004, which is acontinuation-in-part of U.S. patent application Ser. No. 10/780,447,filed Feb. 13, 2004, which is a continuation-in-part of U.S. patentapplication Ser. No. 10/427,160, filed Apr. 30, 2003, which is acontinuation-in-part of International Patent Application No.PCT/US02/15876 filed May 17, 2002, which claims the benefit of U.S.Provisional Application No. 60/292,217, filed May 18, 2001, U.S.Provisional Application No. 60/362,016, filed Mar. 6, 2002, U.S.Provisional Application No. 60/306,883, filed Jul. 20, 2001, and U.S.Provisional Application No. 60/311,865, filed Aug. 13, 2001. Thisapplication is also a continuation-in-part of U.S. patent applicationSer. No. 10/727,780 filed Dec. 3, 2003. This application also claims thebenefit of U.S. Provisional Application No. 60/543,480, filed Feb. 10,2004.

The instant application claims the benefit of all the listedapplications, which are hereby incorporated by reference herein in theirentireties, including the drawings.

FIELD OF THE INVENTION

The present invention relates to compounds, compositions, and methodsfor the study, diagnosis, and treatment of traits, diseases andconditions that respond to the modulation of Stearoyl-CoA desaturase(SCD) gene expression and/or activity. The present invention is alsodirected to compounds, compositions, and methods relating to traits,diseases and conditions that respond to the modulation of expressionand/or activity of genes involved in Stearoyl-CoA desaturase (SCD) geneexpression pathways or other cellular processes that mediate themaintenance or development of such traits, diseases and conditions.Specifically, the invention relates to small nucleic acid molecules,such as short interfering nucleic acid (siNA), short interfering RNA(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and shorthairpin RNA (shRNA) molecules capable of mediating RNA interference(RNAi) against Stearoyl-CoA desaturase (SCD) gene expression. Such smallnucleic acid molecules are useful, for example, in providingcompositions for treatment of traits, diseases and conditions that canrespond to modulation of SCD expression in a subject, such as diabetes(type I and/or type II), atherosclerosis, cancer, obesity, and viralinfection.

BACKGROUND OF THE INVENTION

The following is a discussion of relevant art pertaining to RNAi. Thediscussion is provided only for understanding of the invention thatfollows. The summary is not an admission that any of the work describedbelow is prior art to the claimed invention.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fireet al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286,950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, Genes &Dev., 13:139-141; and Strauss, 1999, Science, 286, 886). Thecorresponding process in plants (Heifetz et al., International PCTPublication No. WO 99/61631) is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla (Fire et al., 1999, TrendsGenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or from the random integration oftransposon elements into a host genome via a cellular response thatspecifically destroys homologous single-stranded RNA or viral genomicRNA. The presence of dsRNA in cells triggers the RNAi response through amechanism that has yet to be fully characterized. This mechanism appearsto be different from other known mechanisms involving double strandedRNA-specific ribonucleases, such as the interferon response that resultsfrom dsRNA-mediated activation of protein kinase PKR and2′,5′-oligoadenylate synthetase resulting in non-specific cleavage ofmRNA by ribonuclease L (see for example U.S. Pat. Nos. 6,107,094;5,898,031; Clemens et al., 1997, J. Interferon & Cytokine Res., 17,503-524; Adah et al., 2001, Curr. Med. Chem., 8, 1189).

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer (Bass, 2000, Cell, 101,235; Zamore et al., 2000, Cell, 101, 25-33; Hammond et al., 2000,Nature, 404, 293). Dicer is involved in the processing of the dsRNA intoshort pieces of dsRNA known as short interfering RNAs (siRNAs) (Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Berstein etal., 2001, Nature, 409, 363). Short interfering RNAs derived from diceractivity are typically about 21 to about 23 nucleotides in length andcomprise about 19 base pair duplexes (Zamore et al., 2000, Cell, 101,25-33; Elbashir et al., 2001, Genes Dev., 15, 188). Dicer has also beenimplicated in the excision of 21- and 22-nucleotide small temporal RNAs(stRNAs) from precursor RNA of conserved structure that are implicatedin translational control (Hutvagner et al., 2001, Science, 293, 834).The RNAi response also features an endonuclease complex, commonlyreferred to as an RNA-induced silencing complex (RISC), which mediatescleavage of single-stranded RNA having sequence complementary to theantisense strand of the siRNA duplex. Cleavage of the target RNA takesplace in the middle of the region complementary to the antisense strandof the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans.Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAimediated by dsRNA in mammalian systems. Hammond et al., 2000, Nature,404, 293, describe RNAi in Drosophila cells transfected with dsRNA.Elbashir et al., 2001, Nature, 411, 494 and Tuschl et al., InternationalPCT Publication No. WO 01/75164, describe RNAi induced by introductionof duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cellsincluding human embryonic kidney and HeLa cells. Recent work inDrosophila embryonic lysates (Elbashir et al., 2001, EMBO J., 20, 6877and Tuschl et al., International PCT Publication No. WO 01/75164) hasrevealed certain requirements for siRNA length, structure, chemicalcomposition, and sequence that are essential to mediate efficient RNAiactivity. These studies have shown that 21-nucleotide siRNA duplexes aremost active when containing 3′-terminal dinucleotide overhangs.Furthermore, complete substitution of one or both siRNA strands with2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity,whereas substitution of the 3′-terminal siRNA overhang nucleotides with2′-deoxy nucleotides (2′-H) was shown to be tolerated. Single mismatchsequences in the center of the siRNA duplex were also shown to abolishRNAi activity. In addition, these studies also indicate that theposition of the cleavage site in the target RNA is defined by the 5′-endof the siRNA guide sequence rather than the 3′-end of the guide sequence(Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicatedthat a 5′-phosphate on the target-complementary strand of a siRNA duplexis required for siRNA activity and that ATP is utilized to maintain the5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309).

Studies have shown that replacing the 3′-terminal nucleotide overhangingsegments of a 21-mer siRNA duplex having two-nucleotide 3′-overhangswith deoxyribonucleotides does not have an adverse effect on RNAiactivity. Replacing up to four nucleotides on each end of the siRNA withdeoxyribonucleotides has been reported to be well tolerated, whereascomplete substitution with deoxyribonucleotides results in no RNAiactivity (Elbashir et al., 2001, EMBO J., 20, 6877 and Tuschl et al.,International PCT Publication No. WO 01/75164). In addition, Elbashir etal., supra, also report that substitution of siRNA with 2′-O-methylnucleotides completely abolishes RNAi activity. Li et al., InternationalPCT Publication No. WO 00/44914, and Beach et al., International PCTPublication No. WO 01/68836 preliminarily suggest that siRNA may includemodifications to either the phosphate-sugar backbone or the nucleosideto include at least one of a nitrogen or sulfur heteroatom, however,neither application postulates to what extent such modifications wouldbe tolerated in siRNA molecules, nor provides any further guidance orexamples of such modified siRNA. Kreutzer et al., Canadian PatentApplication No. 2,359,180, also describe certain chemical modificationsfor use in dsRNA constructs in order to counteract activation ofdouble-stranded RNA-dependent protein kinase PKR, specifically 2′-aminoor 2′-O-methyl nucleotides, and nucleotides containing a 2′-O or 4′-Cmethylene bridge. However, Kreutzer et al. similarly fails to provideexamples or guidance as to what extent these modifications would betolerated in dsRNA molecules.

Parrish et al., 2000, Molecular Cell, 6, 1077-1087, tested certainchemical modifications targeting the unc-22 gene in C. elegans usinglong (>25 nt) siRNA transcripts. The authors describe the introductionof thiophosphate residues into these siRNA transcripts by incorporatingthiophosphate nucleotide analogs with T7 and T3 RNA polymerase andobserved that RNAs with two phosphorothioate modified bases also hadsubstantial decreases in effectiveness as RNAi. Further, Parrish et al.reported that phosphorothioate modification of more than two residuesgreatly destabilized the RNAs in vitro such that interference activitiescould not be assayed. Id. at 1081. The authors also tested certainmodifications at the 2′-position of the nucleotide sugar in the longsiRNA transcripts and found that substituting deoxynucleotides forribonucleotides produced a substantial decrease in interferenceactivity, especially in the case of Uridine to Thymidine and/or Cytidineto deoxy-Cytidine substitutions. Id. In addition, the authors testedcertain base modifications, including substituting, in sense andantisense strands of the siRNA, 4-thiouracil, 5-bromouracil,5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine forguanosine. Whereas 4-thiouracil and 5-bromouracil substitution appearedto be tolerated, Parrish reported that inosine produced a substantialdecrease in interference activity when incorporated in either strand.Parrish also reported that incorporation of 5-iodouracil and3-(aminoallyl)uracil in the antisense strand resulted in a substantialdecrease in RNAi activity as well.

The use of longer dsRNA has been described. For example, Beach et al.,International PCT Publication No. WO 01/68836, describes specificmethods for attenuating gene expression using endogenously-deriveddsRNA. Tuschl et al., International PCT Publication No. WO 01/75164,describe a Drosophila in vitro RNAi system and the use of specific siRNAmolecules for certain functional genomic and certain therapeuticapplications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubtsthat RNAi can be used to cure genetic diseases or viral infection due tothe danger of activating interferon response. Li et al., InternationalPCT Publication No. WO 00/44914, describe the use of specific long (141bp-488 bp) enzymatically synthesized or vector expressed dsRNAs forattenuating the expression of certain target genes. Zernicka-Goetz etal., International PCT Publication No. WO 01/36646, describe certainmethods for inhibiting the expression of particular genes in mammaliancells using certain long (550 bp-714 bp), enzymatically synthesized orvector expressed dsRNA molecules. Fire et al., International PCTPublication No. WO 99/32619, describe particular methods for introducingcertain long dsRNA molecules into cells for use in inhibiting geneexpression in nematodes. Plaetinck et al., International PCT PublicationNo. WO 00/01846, describe certain methods for identifying specific genesresponsible for conferring a particular phenotype in a cell usingspecific long dsRNA molecules. Mello et al., International PCTPublication No. WO 01/29058, describe the identification of specificgenes involved in dsRNA-mediated RNAi. Pachuck et al., International PCTPublication No. WO 00/63364, describe certain long (at least 200nucleotide) dsRNA constructs. Deschamps Depaillette et al.,International PCT Publication No. WO 99/07409, describe specificcompositions consisting of particular dsRNA molecules combined withcertain anti-viral agents. Waterhouse et al., International PCTPublication No. 99/53050 and 1998, PNAS, 95, 13959-13964, describecertain methods for decreasing the phenotypic expression of a nucleicacid in plant cells using certain dsRNAs. Driscoll et al., InternationalPCT Publication No. WO 01/49844, describe specific DNA expressionconstructs for use in facilitating gene silencing in targeted organisms.

Others have reported on various RNAi and gene-silencing systems. Forexample, Parrish et al., 2000, Molecular Cell, 6, 1077-1087, describespecific chemically-modified dsRNA constructs targeting the unc-22 geneof C. elegans. Grossniklaus, International PCT Publication No. WO01/38551, describes certain methods for regulating polycomb geneexpression in plants using certain dsRNAs. Churikov et al.,International PCT Publication No. WO 01/42443, describe certain methodsfor modifying genetic characteristics of an organism using certaindsRNAs. Cogoni et al,, International PCT Publication No. WO 01/53475,describe certain methods for isolating a Neurospora silencing gene anduses thereof. Reed et al., International PCT Publication No. WO01/68836, describe certain methods for gene silencing in plants. Honeret al., International PCT Publication No. WO 01/70944, describe certainmethods of drug screening using transgenic nematodes as Parkinson'sDisease models using certain dsRNAs. Deak et al., International PCTPublication No. WO 01/72774, describe certain Drosophila-derived geneproducts that may be related to RNAi in Drosophila. Arndt et al.,International PCT Publication No. WO 01/92513 describe certain methodsfor mediating gene suppression by using factors that enhance RNAi.Tuschl et al., International PCT Publication No. WO 02/44321, describecertain synthetic siRNA constructs. Pachuk et al., International PCTPublication No. WO 00/63364, and Satishchandran et al., InternationalPCT Publication No. WO 01/04313, describe certain methods andcompositions for inhibiting the function of certain polynucleotidesequences using certain long (over 250 bp), vector expressed dsRNAs.Echeverri et al., International PCT Publication No. WO 02/38805,describe certain C. elegans genes identified via RNAi. Kreutzer et al.,International PCT Publications Nos. WO 02/055692, WO 02/055693, and EP1144623 B1 describes certain methods for inhibiting gene expressionusing dsRNA. Graham et al., International PCT Publications Nos. WO99/49029 and WO 01/70949, and AU 4037501 describe certain vectorexpressed siRNA molecules. Fire et al., U.S. Pat. No. 6,506,559,describe certain methods for inhibiting gene expression in vitro usingcertain long dsRNA (299 bp-1033 bp) constructs that mediate RNAi.Martinez et al., 2002, Cell, 110, 563-574, describe certain singlestranded siRNA constructs, including certain 5′-phosphorylated singlestranded siRNAs that mediate RNA interference in Hela cells. Harborth etal., 2003, Antisense & Nucleic Acid Drug Development, 13, 83-105,describe certain chemically and structurally modified siRNA molecules.Chiu and Rana, 2003, RNA, 9, 1034-1048, describe certain chemically andstructurally modified siRNA molecules. Woolf et al., International PCTPublication Nos. WO 03/064626 and WO 03/064625 describe certainchemically modified dsRNA constructs.

Brownlie et al., International PCT Publication No. WO 01/62954, describemethods and compositions using stearoyl-CoA desaturase to identifycertain triglyceride reducing therapeutic agents.

SUMMARY OF THE INVENTION

This invention relates to compounds, compositions, and methods usefulfor modulating Stearoyl-CoA desaturase (SCD) gene expression using shortinterfering nucleic acid (siNA) molecules. This invention also relatesto compounds, compositions, and methods useful for modulating theexpression and activity of other genes involved in pathways of SCD geneexpression and/or activity by RNA interference (RNAi) using smallnucleic acid molecules. In particular, the instant invention featuressmall nucleic acid molecules, such as short interfering nucleic acid(siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA),micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methodsused to modulate the expression of SCD genes.

A siNA of the invention can be unmodified or chemically-modified. A siNAof the instant invention can be chemically synthesized, expressed from avector or enzymatically synthesized. The instant invention also featuresvarious chemically-modified synthetic short interfering nucleic acid(siNA) molecules capable of modulating SCD gene expression or activityin cells by RNA interference (RNAi). The use of chemically-modified siNAimproves various properties of native siNA molecules through increasedresistance to nuclease degradation in vivo and/or through improvedcellular uptake. Further, contrary to earlier published studies, siNAhaving multiple chemical modifications retains its RNAi activity. ThesiNA molecules of the instant invention provide useful reagents andmethods for a variety of therapeutic, veterinary, diagnostic, targetvalidation, genomic discovery, genetic engineering, and pharmacogenomicapplications.

In one embodiment, the invention features one or more siNA molecules andmethods that independently or in combination modulate the expression ofSCD genes encoding proteins, such as proteins comprising SCD associatedwith the maintenance and/or development of diabetes (type I and/or typeII), atherosclerosis, cancer, obesity, and viral infection, such asgenes encoding sequences comprising those sequences referred to byGenBank Accession Nos. shown in Table I, referred to herein generally asStearoyl-CoA desaturase or SCD. The description below of the variousaspects and embodiments of the invention is provided with reference toexemplary Stearoyl-CoA desaturase gene referred to herein as SCD.However, the various aspects and embodiments are also directed to otherSCD genes, such as homolog genes and transcript variants, andpolymorphisms (e.g., single nucleotide polymorphism, (SNPs)) associatedwith certain SCD genes. As such, the various aspects and embodiments arealso directed to other genes that are involved in SCD mediated pathwaysof signal transduction or gene expression that are involved, forexample, in the maintenance or development of diseases, traits, orconditions described herein. These additional genes can be analyzed fortarget sites using the methods described for SCD genes herein. Thus, themodulation of other genes and the effects of such modulation of theother genes can be performed, determined, and measured as describedherein.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a SCD gene, wherein said siNA molecule comprises about 15 to about 28base pairs.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that directs cleavage of a SCDRNA via RNA interference (RNAi), wherein the double stranded siNAmolecule comprises a first and a second strand, each strand of the siNAmolecule is about 18 to about 28 nucleotides in length, the first strandof the siNA molecule comprises nucleotide sequence having sufficientcomplementarity to the SCD RNA for the siNA molecule to direct cleavageof the SCD RNA via RNA interference, and the second strand of said siNAmolecule comprises nucleotide sequence that is complementary to thefirst strand.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that directs cleavage of a SCDRNA via RNA interference (RNAi), wherein the double stranded siNAmolecule comprises a first and a second strand, each strand of the siNAmolecule is about 18 to about 23 nucleotides in length, the first strandof the siNA molecule comprises nucleotide sequence having sufficientcomplementarity to the SCD RNA for the siNA molecule to direct cleavageof the SCD RNA via RNA interference, and the second strand of said siNAmolecule comprises nucleotide sequence that is complementary to thefirst strand.

In one embodiment, the invention features a chemically synthesizeddouble stranded short interfering nucleic acid (siNA) molecule thatdirects cleavage of a SCD RNA via RNA interference (RNAi), wherein eachstrand of the siNA molecule is about 18 to about 28 nucleotides inlength; and one strand of the siNA molecule comprises nucleotidesequence having sufficient complementarity to the SCD RNA for the siNAmolecule to direct cleavage of the SCD RNA via RNA interference.

In one embodiment, the invention features a chemically synthesizeddouble stranded short interfering nucleic acid (siNA) molecule thatdirects cleavage of a SCD RNA via RNA interference (RNAi), wherein eachstrand of the siNA molecule is about 18 to about 23 nucleotides inlength; and one strand of the siNA molecule comprises nucleotidesequence having sufficient complementarity to the SCD RNA for the siNAmolecule to direct cleavage of the SCD RNA via RNA interference.

In one embodiment, the invention features a siNA molecule thatdown-regulates expression of a SCD gene, for example, wherein the SCDgene comprises SCD encoding sequence. In one embodiment, the inventionfeatures a siNA molecule that down-regulates expression of a SCD gene,for example, wherein the SCD gene comprises SCD non-coding sequence orregulatory elements involved in SCD gene expression.

In one embodiment, a siNA of the invention is used to inhibit theexpression of SCD genes or a SCD gene family, wherein the genes or genefamily sequences share sequence homology. Such homologous sequences canbe identified as is known in the art, for example using sequencealignments. siNA molecules can be designed to target such homologoussequences, for example using perfectly complementary sequences or byincorporating non-canonical base pairs, for example mismatches and/orwobble base pairs, that can provide additional target sequences. Ininstances where mismatches are identified, non-canonical base pairs (forexample, mismatches and/or wobble bases) can be used to generate siNAmolecules that target more than one gene sequence. In a non-limitingexample, non-canonical base pairs such as UU and CC base pairs are usedto generate siNA molecules that are capable of targeting sequences fordiffering SCD targets that share sequence homology. As such, oneadvantage of using siNAs of the invention is that a single siNA can bedesigned to include nucleic acid sequence that is complementary to thenucleotide sequence that is conserved between the homologous genes. Inthis approach, a single siNA can be used to inhibit expression of morethan one gene instead of using more than one siNA molecule to target thedifferent genes.

In one embodiment, the invention features a siNA molecule having RNAiactivity against SCD RNA, wherein the siNA molecule comprises a sequencecomplementary to any RNA having SCD encoding sequence, such as thosesequences having GenBank Accession Nos. shown in Table I. In anotherembodiment, the invention features a siNA molecule having RNAi activityagainst SCD RNA, wherein the siNA molecule comprises a sequencecomplementary to an RNA having variant SCD encoding sequence, forexample other mutant SCD genes not shown in Table I but known in the artto be associated with the maintenance and/or development of diabetes(type I and/or type II), atherosclerosis, cancer, obesity, and viralinfection. Chemical modifications as shown in Tables III and IV orotherwise described herein can be applied to any siNA construct of theinvention. In another embodiment, a siNA molecule of the inventionincludes a nucleotide sequence that can interact with nucleotidesequence of a SCD gene and thereby mediate silencing of SCD geneexpression, for example, wherein the siNA mediates regulation of SCDgene expression by cellular processes that modulate the chromatinstructure or methylation patterns of the SCD gene and preventtranscription of the SCD gene.

In one embodiment, siNA molecules of the invention are used to downregulate or inhibit the expression of SCD proteins arising from SCDhaplotype polymorphisms that are associated with a disease or condition,(e.g., diabetes (type I and/or type II), atherosclerosis, cancer,obesity, and viral infection). Analysis of SCD genes, or SCD protein orRNA levels can be used to identify subjects with such polymorphisms orthose subjects who are at risk of developing traits, conditions, ordiseases described herein. These subjects are amenable to treatment, forexample, treatment with siNA molecules of the invention and any othercomposition useful in treating diseases related to SCD gene expression.As such, analysis of SCD protein or RNA levels can be used to determinetreatment type and the course of therapy in treating a subject.Monitoring of SCD protein or RNA levels can be used to predict treatmentoutcome and to determine the efficacy of compounds and compositions thatmodulate the level and/or activity of certain SCD proteins associatedwith a trait, condition, or disease.

In one embodiment of the invention a siNA molecule comprises anantisense strand comprising a nucleotide sequence that is complementaryto a nucleotide sequence or a portion thereof encoding a SCD protein.The siNA further comprises a sense strand, wherein said sense strandcomprises a nucleotide sequence of a SCD gene or a portion thereof.

In another embodiment, a siNA molecule comprises an antisense regioncomprising a nucleotide sequence that is complementary to a nucleotidesequence encoding a SCD protein or a portion thereof. The siNA moleculefurther comprises a sense region, wherein said sense region comprises anucleotide sequence of a SCD gene or a portion thereof.

In another embodiment, the invention features a siNA molecule comprisinga nucleotide sequence in the antisense region of the siNA molecule thatis complementary to a nucleotide sequence or portion of sequence of aSCD gene. In another embodiment, the invention features a siNA moleculecomprising a region, for example, the antisense region of the siNAconstruct, complementary to a sequence comprising a SCD gene sequence ora portion thereof.

In one embodiment, the antisense region of SCD siNA constructs comprisesa sequence complementary to sequence having any of SEQ ID NOs. 1-290 or581-596. In one embodiment, the antisense region of SCD constructscomprises sequence having any of SEQ ID NOs. 291-580, 605-612, 621-628,637-644, 653-660, 669-692, 701-708, 717-724, 733-740, 749-756, 765-788,790, 792, 794, 797, 799, 801, 803, or 806. In another embodiment, thesense region of SCD constructs comprises sequence having any of SEQ IDNOs. 1-290, 581-604, 613-620, 629-636, 645-652, 661-668, 693-700,709-716, 725-732, 741-748, 757-764, 789, 791, 793, 795, 796, 798, 800,802, 804, or 805.

In one embodiment, a siNA molecule of the invention comprises any of SEQID NOs. 1-806. The sequences shown in SEQ ID NOs: 1-806 are notlimiting. A siNA molecule of the invention can comprise any contiguousSCD sequence (e.g., about 15 to about 25 or more, or about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, or 25 or more contiguous SCD nucleotides).

In yet another embodiment, the invention features a siNA moleculecomprising a sequence, for example, the antisense sequence of the siNAconstruct, complementary to a sequence or portion of sequence comprisingsequence represented by GenBank Accession Nos. shown in Table I.Chemical modifications in Tables III and IV and described herein can beapplied to any siNA construct of the invention.

In one embodiment of the invention a siNA molecule comprises anantisense strand having about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides,wherein the antisense strand is complementary to a RNA sequence or aportion thereof encoding a SCD protein, and wherein said siNA furthercomprises a sense strand having about 15 to about 30 (e.g., about 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides, and wherein said sense strand and said antisense strand aredistinct nucleotide sequences where at least about 15 nucleotides ineach strand are complementary to the other strand.

In another embodiment of the invention a siNA molecule of the inventioncomprises an antisense region having about 15 to about 30 (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides, wherein the antisense region is complementary to a RNAsequence encoding a SCD protein, and wherein said siNA further comprisesa sense region having about 15 to about 30 (e.g., about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, whereinsaid sense region and said antisense region are comprised in a linearmolecule where the sense region comprises at least about 15 nucleotidesthat are complementary to the antisense region.

In one embodiment, a siNA molecule of the invention has RNAi activitythat modulates expression of RNA encoded by a SCD gene. Because SCDgenes can share some degree of sequence homology with each other, siNAmolecules can be designed to target a class of SCD genes or alternatelyspecific SCD genes (e.g., polymorphic variants) by selecting sequencesthat are either shared amongst different SCD targets or alternativelythat are unique for a specific SCD target. Therefore, in one embodiment,the siNA molecule can be designed to target conserved regions of SCD RNAsequences having homology among several SCD gene variants so as totarget a class of SCD genes with one siNA molecule. Accordingly, in oneembodiment, the siNA molecule of the invention modulates the expressionof one or both SCD alleles in a subject. In another embodiment, the siNAmolecule can be designed to target a sequence that is unique to aspecific SCD RNA sequence (e.g., a single SCD allele or SCD singlenucleotide polymorphism (SNP)) due to the high degree of specificitythat the siNA molecule requires to mediate RNAi activity.

In one embodiment, nucleic acid molecules of the invention that act asmediators of the RNA interference gene silencing response aredouble-stranded nucleic acid molecules. In another embodiment, the siNAmolecules of the invention consist of duplex nucleic acid moleculescontaining about 15 to about 30 base pairs between oligonucleotidescomprising about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In yet anotherembodiment, siNA molecules of the invention comprise duplex nucleic acidmolecules with overhanging ends of about 1 to about 3 (e.g., about 1, 2,or 3) nucleotides, for example, about 21-nucleotide duplexes with about19 base pairs and 3′-terminal mononucleotide, dinucleotide, ortrinucleotide overhangs. In yet another embodiment, siNA molecules ofthe invention comprise duplex nucleic acid molecules with blunt ends,where both ends are blunt, or alternatively, where one of the ends isblunt.

In one embodiment, the invention features one or morechemically-modified siNA constructs having specificity for SCDexpressing nucleic acid molecules, such as RNA encoding a SCD protein.In one embodiment, the invention features a RNA based siNA molecule(e.g., a siNA comprising 2′-OH nucleotides) having specificity for SCDexpressing nucleic acid molecules that includes one or more chemicalmodifications described herein. Non-limiting examples of such chemicalmodifications include without limitation phosphorothioateinternucleotide linkages, 2′-deoxyribonucleotides, 2′-O-methylribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, “universal base”nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, and terminalglyceryl and/or inverted deoxy abasic residue incorporation. Thesechemical modifications, when used in various siNA constructs, (e.g., RNAbased siNA constructs), are shown to preserve RNAi activity in cellswhile at the same time, dramatically increasing the serum stability ofthese compounds. Furthermore, contrary to the data published by Parrishet al., supra, applicant demonstrates that multiple (greater than one)phosphorothioate substitutions are well-tolerated and confer substantialincreases in serum stability for modified siNA constructs.

In one embodiment, a siNA molecule of the invention comprises modifiednucleotides while maintaining the ability to mediate RNAi. The modifiednucleotides can be used to improve in vitro or in vivo characteristicssuch as stability, activity, and/or bioavailability. For example, a siNAmolecule of the invention can comprise modified nucleotides as apercentage of the total number of nucleotides present in the siNAmolecule. As such, a siNA molecule of the invention can generallycomprise about 5% to about 100% modified nucleotides (e.g., about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or 100% modified nucleotides). The actual percentageof modified nucleotides present in a given siNA molecule will depend onthe total number of nucleotides present in the siNA. If the siNAmolecule is single stranded, the percent modification can be based uponthe total number of nucleotides present in the single stranded siNAmolecules. Likewise, if the siNA molecule is double stranded, thepercent modification can be based upon the total number of nucleotidespresent in the sense strand, antisense strand, or both the sense andantisense strands.

One aspect of the invention features a double-stranded short interferingnucleic acid (siNA) molecule that down-regulates expression of a SCDgene. In one embodiment, the double stranded siNA molecule comprises oneor more chemical modifications and each strand of the double-strandedsiNA is about 21 nucleotides long. In one embodiment, thedouble-stranded siNA molecule does not contain any ribonucleotides. Inanother embodiment, the double-stranded siNA molecule comprises one ormore ribonucleotides. In one embodiment, each strand of thedouble-stranded siNA molecule independently comprises about 15 to about30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30) nucleotides, wherein each strand comprises about 15 to about30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30) nucleotides that are complementary to the nucleotides of theother strand. In one embodiment, one of the strands of thedouble-stranded siNA molecule comprises a nucleotide sequence that iscomplementary to a nucleotide sequence or a portion thereof of the SCDgene, and the second strand of the double-stranded siNA moleculecomprises a nucleotide sequence substantially similar to the nucleotidesequence of the SCD gene or a portion thereof.

In another embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a SCD gene comprising an antisense region, wherein the antisenseregion comprises a nucleotide sequence that is complementary to anucleotide sequence of the SCD gene or a portion thereof, and a senseregion, wherein the sense region comprises a nucleotide sequencesubstantially similar to the nucleotide sequence of the SCD gene or aportion thereof. In one embodiment, the antisense region and the senseregion independently comprise about 15 to about 30 (e.g. about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides,wherein the antisense region comprises about 15 to about 30 (e.g. about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides that are complementary to nucleotides of the sense region.

In another embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a SCD gene comprising a sense region and an antisense region, whereinthe antisense region comprises a nucleotide sequence that iscomplementary to a nucleotide sequence of RNA encoded by the SCD gene ora portion thereof and the sense region comprises a nucleotide sequencethat is complementary to the antisense region.

In one embodiment, a siNA molecule of the invention comprises bluntends, i.e., ends that do not include any overhanging nucleotides. Forexample, a siNA molecule comprising modifications described herein(e.g., comprising nucleotides having Formulae I-VII or siNA constructscomprising “Stab 00”-“Stab 32” (Table IV) or any combination thereof(see Table IV)) and/or any length described herein can comprise bluntends or ends with no overhanging nucleotides.

In one embodiment, any siNA molecule of the invention can comprise oneor more blunt ends, i.e. where a blunt end does not have any overhangingnucleotides. In one embodiment, the blunt ended siNA molecule has anumber of base pairs equal to the number of nucleotides present in eachstrand of the siNA molecule. In another embodiment, the siNA moleculecomprises one blunt end, for example wherein the 5′-end of the antisensestrand and the 3′-end of the sense strand do not have any overhangingnucleotides. In another example, the siNA molecule comprises one bluntend, for example wherein the 3′-end of the antisense strand and the5′-end of the sense strand do not have any overhanging nucleotides. Inanother example, a siNA molecule comprises two blunt ends, for examplewherein the 3′-end of the antisense strand and the 5′-end of the sensestrand as well as the 5′-end of the antisense strand and 3′-end of thesense strand do not have any overhanging nucleotides. A blunt ended siNAmolecule can comprise, for example, from about 15 to about 30nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 nucleotides). Other nucleotides present in a bluntended siNA molecule can comprise, for example, mismatches, bulges,loops, or wobble base pairs to modulate the activity of the siNAmolecule to mediate RNA interference.

By “blunt ends” is meant symmetric termini or termini of a doublestranded siNA molecule having no overhanging nucleotides. The twostrands of a double stranded siNA molecule align with each other withoutover-hanging nucleotides at the termini. For example, a blunt ended siNAconstruct comprises terminal nucleotides that are complementary betweenthe sense and antisense regions of the siNA molecule.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a SCD gene, wherein the siNA molecule is assembled from two separateoligonucleotide fragments wherein one fragment comprises the senseregion and the second fragment comprises the antisense region of thesiNA molecule. The sense region can be connected to the antisense regionvia a linker molecule, such as a polynucleotide linker or anon-nucleotide linker.

In one embodiment, the invention features double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a SCD gene, wherein the siNA molecule comprises about 15 to about 30(e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30) base pairs, and wherein each strand of the siNA moleculecomprises one or more chemical modifications. In another embodiment, oneof the strands of the double-stranded siNA molecule comprises anucleotide sequence that is complementary to a nucleotide sequence of aSCD gene or a portion thereof, and the second strand of thedouble-stranded siNA molecule comprises a nucleotide sequencesubstantially similar to the nucleotide sequence or a portion thereof ofthe SCD gene. In another embodiment, one of the strands of thedouble-stranded siNA molecule comprises a nucleotide sequence that iscomplementary to a nucleotide sequence of a SCD gene or portion thereof,and the second strand of the double-stranded siNA molecule comprises anucleotide sequence substantially similar to the nucleotide sequence orportion thereof of the SCD gene. In another embodiment, each strand ofthe siNA molecule comprises about 15 to about 30 (e.g. about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, andeach strand comprises at least about 15 to about 30 (e.g. about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotidesthat are complementary to the nucleotides of the other strand. The SCDgene can comprise, for example, sequences referred to in Table I.

In one embodiment, a siNA molecule of the invention comprises noribonucleotides. In another embodiment, a siNA molecule of the inventioncomprises ribonucleotides.

In one embodiment, a siNA molecule of the invention comprises anantisense region comprising a nucleotide sequence that is complementaryto a nucleotide sequence of a SCD gene or a portion thereof, and thesiNA further comprises a sense region comprising a nucleotide sequencesubstantially similar to the nucleotide sequence of the SCD gene or aportion thereof. In another embodiment, the antisense region and thesense region each comprise about 15 to about 30 (e.g. about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides andthe antisense region comprises at least about 15 to about 30 (e.g. about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides that are complementary to nucleotides of the sense region.The SCD gene can comprise, for example, sequences referred to in TableI. In another embodiment, the siNA is a double stranded nucleic acidmolecule, where each of the two strands of the siNA moleculeindependently comprise about 15 to about 40 (e.g. about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36,37, 38, 39, or 40) nucleotides, and where one of the strands of the siNAmolecule comprises at least about 15 (e.g. about 15, 16, 17, 18, 19, 20,21, 22, 23, 24 or 25 or more) nucleotides that are complementary to thenucleic acid sequence of the SCD gene or a portion thereof.

In one embodiment, a siNA molecule of the invention comprises a senseregion and an antisense region, wherein the antisense region comprises anucleotide sequence that is complementary to a nucleotide sequence ofRNA encoded by a SCD gene, or a portion thereof, and the sense regioncomprises a nucleotide sequence that is complementary to the antisenseregion. In one embodiment, the siNA molecule is assembled from twoseparate oligonucleotide fragments, wherein one fragment comprises thesense region and the second fragment comprises the antisense region ofthe siNA molecule. In another embodiment, the sense region is connectedto the antisense region via a linker molecule. In another embodiment,the sense region is connected to the antisense region via a linkermolecule, such as a nucleotide or non-nucleotide linker. The SCD genecan comprise, for example, sequences referred in to Table I.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a SCD gene comprising a sense region and an antisense region, whereinthe antisense region comprises a nucleotide sequence that iscomplementary to a nucleotide sequence of RNA encoded by the SCD gene ora portion thereof and the sense region comprises a nucleotide sequencethat is complementary to the antisense region, and wherein the siNAmolecule has one or more modified pyrimidine and/or purine nucleotides.In one embodiment, the pyrimidine nucleotides in the sense region are2′-O-methyl pyrimidine nucleotides or 2′-deoxy-2′-fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-deoxy purine nucleotides. In another embodiment, the pyrimidinenucleotides in the sense region are 2′-deoxy-2′-fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-O-methyl purine nucleotides. In another embodiment, the pyrimidinenucleotides in the sense region are 2′-deoxy-2′-fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-deoxy purine nucleotides. In one embodiment, the pyrimidinenucleotides in the antisense region are 2′-deoxy-2′-fluoro pyrimidinenucleotides and the purine nucleotides present in the antisense regionare 2′-O-methyl or 2′-deoxy purine nucleotides. In another embodiment ofany of the above-described siNA molecules, any nucleotides present in anon-complementary region of the sense strand (e.g. overhang region) are2′-deoxy nucleotides.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a SCD gene, wherein the siNA molecule is assembled from two separateoligonucleotide fragments wherein one fragment comprises the senseregion and the second fragment comprises the antisense region of thesiNA molecule, and wherein the fragment comprising the sense regionincludes a terminal cap moiety at the 5′-end, the 3′-end, or both of the5′ and 3′ ends of the fragment. In one embodiment, the terminal capmoiety is an inverted deoxy abasic moiety or glyceryl moiety. In oneembodiment, each of the two fragments of the siNA molecule independentlycomprise about 15 to about 30 (e.g. about 15, 16,-17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In anotherembodiment, each of the two fragments of the siNA molecule independentlycomprise about 15 to about 40 (e.g. about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39,or 40) nucleotides. In a non-limiting example, each of the two fragmentsof the siNA molecule comprise about 21 nucleotides.

In one embodiment, the invention features a siNA molecule comprising atleast one modified nucleotide, wherein the modified nucleotide is a2′-deoxy-2′-fluoro nucleotide. The siNA can be, for example, about 15 toabout 40 nucleotides in length. In one embodiment, all pyrimidinenucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidinenucleotides. In one embodiment, the modified nucleotides in the siNAinclude at least one 2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluorouridine nucleotide. In another embodiment, the modified nucleotides inthe siNA include at least one 2′-fluoro cytidine and at least one2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all uridinenucleotides present in the siNA are 2′-deoxy-2′-fluoro uridinenucleotides. In one embodiment, all cytidine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro cytidine nucleotides. In one embodiment, alladenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroadenosine nucleotides. In one embodiment, all guanosine nucleotidespresent in the siNA are 2′-deoxy-2′-fluoro guanosine nucleotides. ThesiNA can further comprise at least one modified internucleotidiclinkage, such as phosphorothioate linkage. In one embodiment, the2′-deoxy-2′-fluoronucleotides are present at specifically selectedlocations in the siNA that are sensitive to cleavage by ribonucleases,such as locations having pyrimidine nucleotides.

In one embodiment, the invention features a method of increasing thestability of a siNA molecule against cleavage by ribonucleasescomprising introducing at least one modified nucleotide into the siNAmolecule, wherein the modified nucleotide is a 2′-deoxy-2′-fluoronucleotide. In one embodiment, all pyrimidine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In one embodiment,the modified nucleotides in the siNA include at least one2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluoro uridine nucleotide. Inanother embodiment, the modified nucleotides in the siNA include atleast one 2′-fluoro cytidine and at least one 2′-deoxy-2′-fluoro uridinenucleotides. In one embodiment, all uridine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, allcytidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro cytidinenucleotides. In one embodiment, all adenosine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro adenosine nucleotides. In one embodiment,all guanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroguanosine nucleotides. The siNA can further comprise at least onemodified internucleotidic linkage, such as phosphorothioate linkage. Inone embodiment, the 2′-deoxy-2′-fluoronucleotides are present atspecifically selected locations in the siNA that are sensitive tocleavage by ribonucleases, such as locations having pyrimidinenucleotides.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a SCD gene comprising a sense region and an antisense region, whereinthe antisense region comprises a nucleotide sequence that iscomplementary to a nucleotide sequence of RNA encoded by the SCD gene ora portion thereof and the sense region comprises a nucleotide sequencethat is complementary to the antisense region, and wherein the purinenucleotides present in the antisense region comprise 2′-deoxy- purinenucleotides. In an alternative embodiment, the purine nucleotidespresent in the antisense region comprise 2′-O-methyl purine nucleotides.In either of the above embodiments, the antisense region can comprise aphosphorothioate internucleotide linkage at the 3′ end of the antisenseregion. Alternatively, in either of the above embodiments, the antisenseregion can comprise a glyceryl modification at the 3′ end of theantisense region. In another embodiment of any of the above-describedsiNA molecules, any nucleotides present in a non-complementary region ofthe antisense strand (e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the antisense region of a siNA molecule of theinvention comprises sequence complementary to a portion of a SCDtranscript having sequence unique to a particular SCD disease relatedallele, such as sequence comprising a single nucleotide polymorphism(SNP) associated with the disease specific allele. As such, theantisense region of a siNA molecule of the invention can comprisesequence complementary to sequences that are unique to a particularallele to provide specificity in mediating selective RNAi against thedisease, condition, or trait related allele.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a SCD gene, wherein the siNA molecule is assembled from two separateoligonucleotide fragments wherein one fragment comprises the senseregion and the second fragment comprises the antisense region of thesiNA molecule. In another embodiment, the siNA molecule is a doublestranded nucleic acid molecule, where each strand is about 21nucleotides long and where about 19 nucleotides of each fragment of thesiNA molecule are base-paired to the complementary nucleotides of theother fragment of the siNA molecule, wherein at least two 3′ terminalnucleotides of each fragment of the siNA molecule are not base-paired tothe nucleotides of the other fragment of the siNA molecule. In anotherembodiment, the siNA molecule is a double stranded nucleic acidmolecule, where each strand is about 19 nucleotide long and where thenucleotides of each fragment of the siNA molecule are base-paired to thecomplementary nucleotides of the other fragment of the siNA molecule toform at least about 15 (e.g., 15, 16, 17, 18, or 19) base pairs, whereinone or both ends of the siNA molecule are blunt ends. In one embodiment,each of the two 3′ terminal nucleotides of each fragment of the siNAmolecule is a 2′-deoxy-pyrimidine nucleotide, such as a2′-deoxy-thymidine. In another embodiment, all nucleotides of eachfragment of the siNA molecule are base-paired to the complementarynucleotides of the other fragment of the siNA molecule. In anotherembodiment, the siNA molecule is a double stranded nucleic acid moleculeof about 19 to about 25 base pairs having a sense region and anantisense region, where about 19 nucleotides of the antisense region arebase-paired to the nucleotide sequence or a portion thereof of the RNAencoded by the SCD gene. In another embodiment, about 21 nucleotides ofthe antisense region are base-paired to the nucleotide sequence or aportion thereof of the RNA encoded by the SCD gene. In any of the aboveembodiments, the 5′-end of the fragment comprising said antisense regioncan optionally include a phosphate group.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits the expression ofa SCD RNA sequence (e.g., wherein said target RNA sequence is encoded bya SCD gene involved in the SCD pathway), wherein the siNA molecule doesnot contain any ribonucleotides and wherein each strand of thedouble-stranded siNA molecule is about 15 to about 30 nucleotides. Inone embodiment, the siNA molecule is 21 nucleotides in length. Examplesof non-ribonucleotide containing siNA constructs are combinations ofstabilization chemistries shown in Table IV in any combination ofSense/Antisense chemistries, such as Stab 7/8, Stab 7/11, Stab 8/8, Stab18/8, Stab 18/11, Stab 12/13, Stab 7/13, Stab 18/13, Stab 7/19, Stab8/19, Stab 18/19, Stab 7/20, Stab 8/20, Stab 18/20, Stab 7/32, Stab8/32, or Stab 18/32 (e.g., any siNA having Stab 7, 8, 11, 12, 13, 14,15, 17, 18, 19, 20, or 32 sense or antisense strands or any combinationthereof).

In one embodiment, the invention features a chemically synthesizeddouble stranded RNA molecule that directs cleavage of a SCD RNA via RNAinterference, wherein each strand of said RNA molecule is about 15 toabout 30 nucleotides in length; one strand of the RNA molecule comprisesnucleotide sequence having sufficient complementarity to the SCD RNA forthe RNA molecule to direct cleavage of the SCD RNA via RNA interference;and wherein at least one strand of the RNA molecule optionally comprisesone or more chemically modified nucleotides described herein, such aswithout limitation deoxynucleotides, 2′-O-methyl nucleotides,2′-deoxy-2′-fluoro nucleotides, 2′-O-methoxyethyl nucleotides etc.

In one embodiment, the invention features a medicament comprising a siNAmolecule of the invention.

In one embodiment, the invention features an active ingredientcomprising a siNA molecule of the invention.

In one embodiment, the invention features the use of a double-strandedshort interfering nucleic acid (siNA) molecule to inhibit,down-regulate, or reduce expression of a SCD gene, wherein the siNAmolecule comprises one or more chemical modifications and each strand ofthe double-stranded siNA is independently about 15 to about 30 or more(e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29or 30 or more) nucleotides long. In one embodiment, the siNA molecule ofthe invention is a double stranded nucleic acid molecule comprising oneor more chemical modifications, where each of the two fragments of thesiNA molecule independently comprise about 15 to about 40 (e.g. about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23,33, 34, 35, 36, 37, 38, 39, or 40) nucleotides and where one of thestrands comprises at least 15 nucleotides that are complementary tonucleotide sequence of SCD encoding RNA or a portion thereof. In anon-limiting example, each of the two fragments of the siNA moleculecomprise about 21 nucleotides. In another embodiment, the siNA moleculeis a double stranded nucleic acid molecule comprising one or morechemical modifications, where each strand is about 21 nucleotide longand where about 19 nucleotides of each fragment of the siNA molecule arebase-paired to the complementary nucleotides of the other fragment ofthe siNA molecule, wherein at least two 3′ terminal nucleotides of eachfragment of the siNA molecule are not base-paired to the nucleotides ofthe other fragment of the siNA molecule. In another embodiment, the siNAmolecule is a double stranded nucleic acid molecule comprising one ormore chemical modifications, where each strand is about 19 nucleotidelong and where the nucleotides of each fragment of the siNA molecule arebase-paired to the complementary nucleotides of the other fragment ofthe siNA molecule to form at least about 15 (e.g., 15, 16, 17, 18, or19) base pairs, wherein one or both ends of the siNA molecule are bluntends. In one embodiment, each of the two 3′ terminal nucleotides of eachfragment of the siNA molecule is a 2′-deoxy-pyrimidine nucleotide, suchas a 2′-deoxy-thymidine. In another embodiment, all nucleotides of eachfragment of the siNA molecule are base-paired to the complementarynucleotides of the other fragment of the siNA molecule. In anotherembodiment, the siNA molecule is a double stranded nucleic acid moleculeof about 19 to about 25 base pairs having a sense region and anantisense region and comprising one or more chemical modifications,where about 19 nucleotides of the antisense region are base-paired tothe nucleotide sequence or a portion thereof of the RNA encoded by theSCD gene. In another embodiment, about 21 nucleotides of the antisenseregion are base-paired to the nucleotide sequence or a portion thereofof the RNA encoded by the SCD gene. In any of the above embodiments, the5′-end of the fragment comprising said antisense region can optionallyinclude a phosphate group.

In one embodiment, the invention features the use of a double-strandedshort interfering nucleic acid (siNA) molecule that inhibits,down-regulates, or reduces expression of a SCD gene, wherein one of thestrands of the double-stranded siNA molecule is an antisense strandwhich comprises nucleotide sequence that is complementary to nucleotidesequence of SCD RNA or a portion thereof, the other strand is a sensestrand which comprises nucleotide sequence that is complementary to anucleotide sequence of the antisense strand and wherein a majority ofthe pyrimidine nucleotides present in the double-stranded siNA moleculecomprises a sugar modification.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits, down-regulates,or reduces expression of a SCD gene, wherein one of the strands of thedouble-stranded siNA molecule is an antisense strand which comprisesnucleotide sequence that is complementary to nucleotide sequence of SCDRNA or a portion thereof, wherein the other strand is a sense strandwhich comprises nucleotide sequence that is complementary to anucleotide sequence of the antisense strand and wherein a majority ofthe pyrimidine nucleotides present in the double-stranded siNA moleculecomprises a sugar modification.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits, down-regulates,or reduces expression of a SCD gene, wherein one of the strands of thedouble-stranded siNA molecule is an antisense strand which comprisesnucleotide sequence that is complementary to nucleotide sequence of SCDRNA that encodes a protein or portion thereof, the other strand is asense strand which comprises nucleotide sequence that is complementaryto a nucleotide sequence of the antisense strand and wherein a majorityof the pyrimidine nucleotides present in the double-stranded siNAmolecule comprises a sugar modification. In one embodiment, each strandof the siNA molecule comprises about 15 to about 30 or more (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ormore) nucleotides, wherein each strand comprises at least about 15nucleotides that are complementary to the nucleotides of the otherstrand. In one embodiment, the siNA molecule is assembled from twooligonucleotide fragments, wherein one fragment comprises the nucleotidesequence of the antisense strand of the siNA molecule and a secondfragment comprises nucleotide sequence of the sense region of the siNAmolecule. In one embodiment, the sense strand is connected to theantisense strand via a linker molecule, such as a polynucleotide linkeror a non-nucleotide linker. In a further embodiment, the pyrimidinenucleotides present in the sense strand are 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in the senseregion are 2′-deoxy purine nucleotides. In another embodiment, thepyrimidine nucleotides present in the sense strand are2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotidespresent in the sense region are 2′-O-methyl purine nucleotides. In stillanother embodiment, the pyrimidine nucleotides present in the antisensestrand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and any purinenucleotides present in the antisense strand are 2′-deoxy purinenucleotides. In another embodiment, the antisense strand comprises oneor more 2′-deoxy-2′-fluoro pyrimidine nucleotides and one or more2′-O-methyl purine nucleotides. In another embodiment, the pyrimidinenucleotides present in the antisense strand are 2′-deoxy-2′-fluoropyrimidine nucleotides and any purine nucleotides present in theantisense strand are 2′-O-methyl purine nucleotides. In a furtherembodiment the sense strand comprises a 3′-end and a 5′-end, wherein aterminal cap moiety (e.g., an inverted deoxy abasic moiety or inverteddeoxy nucleotide moiety such as inverted thymidine) is present at the5′-end, the 3′-end, or both of the 5′ and 3′ ends of the sense strand.In another embodiment, the antisense strand comprises a phosphorothioateinternucleotide linkage at the 3′ end of the antisense strand. Inanother embodiment, the antisense strand comprises a glycerylmodification at the 3′ end. In another embodiment, the 5′-end of theantisense strand optionally includes a phosphate group.

In any of the above-described embodiments of a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aSCD gene, wherein a majority of the pyrimidine nucleotides present inthe double-stranded siNA molecule comprises a sugar modification, eachof the two strands of the siNA molecule can comprise about 15 to about30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 or more) nucleotides. In one embodiment, about 15 toabout 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 or more) nucleotides of each strand of thesiNA molecule are base-paired to the complementary nucleotides of theother strand of the siNA molecule. In another embodiment, about 15 toabout 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 or more) nucleotides of each strand of thesiNA molecule are base-paired to the complementary nucleotides of theother strand of the siNA molecule, wherein at least two 3′ terminalnucleotides of each strand of the siNA molecule are not base-paired tothe nucleotides of the other strand of the siNA molecule. In anotherembodiment, each of the two 3′ terminal nucleotides of each fragment ofthe siNA molecule is a 2′-deoxy-pyrimidine, such as 2′-deoxy-thymidine.In one embodiment, each strand of the siNA molecule is base-paired tothe complementary nucleotides of the other strand of the siNA molecule.In one embodiment, about 15 to about 30 (e.g., about 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides of theantisense strand are base-paired to the nucleotide sequence of the SCDRNA or a portion thereof. In one embodiment, about 18 to about 25 (e.g.,about 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides of the antisensestrand are base-paired to the nucleotide sequence of the SCD RNA or aportion thereof.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aSCD gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of SCD RNA or a portion thereof,the other strand is a sense strand which comprises nucleotide sequencethat is complementary to a nucleotide sequence of the antisense strandand wherein a majority of the pyrimidine nucleotides present in thedouble-stranded siNA molecule comprises a sugar modification, andwherein the 5′-end of the antisense strand optionally includes aphosphate group.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aSCD gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of SCD RNA or a portion thereof,the other strand is a sense strand which comprises nucleotide sequencethat is complementary to a nucleotide sequence of the antisense strandand wherein a majority of the pyrimidine nucleotides present in thedouble-stranded siNA molecule comprises a sugar modification, andwherein the nucleotide sequence or a portion thereof of the antisensestrand is complementary to a nucleotide sequence of the untranslatedregion or a portion thereof of the SCD RNA.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aSCD gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of SCD RNA or a portion thereof,wherein the other strand is a sense strand which comprises nucleotidesequence that is complementary to a nucleotide sequence of the antisensestrand, wherein a majority of the pyrimidine nucleotides present in thedouble-stranded siNA molecule comprises a sugar modification, andwherein the nucleotide sequence of the antisense strand is complementaryto a nucleotide sequence of the SCD RNA or a portion thereof that ispresent in the SCD RNA.

In one embodiment, the invention features a composition comprising asiNA molecule of the invention in a pharmaceutically acceptable carrieror diluent.

In a non-limiting example, the introduction of chemically-modifiednucleotides into nucleic acid molecules provides a powerful tool inovercoming potential limitations of in vivo stability andbioavailability inherent to native RNA molecules that are deliveredexogenously. For example, the use of chemically-modified nucleic acidmolecules can enable a lower dose of a particular nucleic acid moleculefor a given therapeutic effect since chemically-modified nucleic acidmolecules tend to have a longer half-life in serum. Furthermore, certainchemical modifications can improve the bioavailability of nucleic acidmolecules by targeting particular cells or tissues and/or improvingcellular uptake of the nucleic acid molecule. Therefore, even if theactivity of a chemically-modified nucleic acid molecule is reduced ascompared to a native nucleic acid molecule, for example, when comparedto an all-RNA nucleic acid molecule, the overall activity of themodified nucleic acid molecule can be greater than that of the nativemolecule due to improved stability and/or delivery of the molecule.Unlike native unmodified siNA, chemically-modified siNA can alsominimize the possibility of activating interferon activity in humans.

In any of the embodiments of siNA molecules described herein, theantisense region of a siNA molecule of the invention can comprise aphosphorothioate internucleotide linkage at the 3′-end of said antisenseregion. In any of the embodiments of siNA molecules described herein,the antisense region can comprise about one to about fivephosphorothioate internucleotide linkages at the 5′-end of saidantisense region. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs of a siNA molecule of theinvention can comprise ribonucleotides or deoxyribonucleotides that arechemically-modified at a nucleic acid sugar, base, or backbone. In anyof the embodiments of siNA molecules described herein, the 3′-terminalnucleotide overhangs can comprise one or more universal baseribonucleotides. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs can comprise one or moreacyclic nucleotides.

One embodiment of the invention provides an expression vector comprisinga nucleic acid sequence encoding at least one siNA molecule of theinvention in a manner that allows expression of the nucleic acidmolecule. Another embodiment of the invention provides a mammalian cellcomprising such an expression vector. The mammalian cell can be a humancell. The siNA molecule of the expression vector can comprise a senseregion and an antisense region. The antisense region can comprisesequence complementary to a RNA or DNA sequence encoding SCD and thesense region can comprise sequence complementary to the antisenseregion. The siNA molecule can comprise two distinct strands havingcomplementary sense and antisense regions. The siNA molecule cancomprise a single strand having complementary sense and antisenseregions.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against SCD inside a cell or reconstituted in vitrosystem, wherein the chemical modification comprises one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides comprising abackbone modified internucleotide linkage having Formula I:

-   -   wherein each R1 and R2 is independently any nucleotide,        non-nucleotide, or polynucleotide which can be        naturally-occurring or chemically-modified, each X and Y is        independently O, S, N, alkyl, or substituted alkyl, each Z and W        is independently O, S, N, alkyl, substituted alkyl, O-alkyl,        S-alkyl, alkaryl, aralkyl, or acetyl and wherein W, X, Y, and Z        are optionally not all O. In another embodiment, a backbone        modification of the invention comprises a phosphonoacetate        and/or thiophosphonoacetate internucleotide linkage (see for        example Sheehan et al., 2003, Nucleic Acids Research, 31,        4109-4118).

The chemically-modified internucleotide linkages having Formula I, forexample, wherein any Z, W, X, and/or Y independently comprises a sulphuratom, can be present in one or both oligonucleotide strands of the siNAduplex, for example, in the sense strand, the antisense strand, or bothstrands. The siNA molecules of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically-modifiedinternucleotide linkages having Formula I at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of the sense strand, the antisense strand, orboth strands. For example, an exemplary siNA molecule of the inventioncan comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, ormore) chemically-modified internucleotide linkages having Formula I atthe 5′-end of the sense strand, the antisense strand, or both strands.In another non-limiting example, an exemplary siNA molecule of theinvention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) pyrimidine nucleotides with chemically-modifiedinternucleotide linkages having Formula I in the sense strand, theantisense strand, or both strands. In yet another non-limiting example,an exemplary siNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotideswith chemically-modified internucleotide linkages having Formula I inthe sense strand, the antisense strand, or both strands. In anotherembodiment, a siNA molecule of the invention having internucleotidelinkage(s) of Formula I also comprises a chemically-modified nucleotideor non-nucleotide having any of Formulae I-VII.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against SCD inside a cell or reconstituted in vitrosystem, wherein the chemical modification comprises one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides ornon-nucleotides having Formula II:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalklylamino, substituted silyl, or group having Formula I or II; R9is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, or any other non-naturallyoccurring base that can be complementary or non-complementary to targetRNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole,5-nitroindole, nebularine, pyridone, pyridinone, or any othernon-naturally occurring universal base that can be complementary ornon-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Formula II canbe present in one or both oligonucleotide strands of the siNA duplex,for example in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or morechemically-modified nucleotides or non-nucleotides of Formula II at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand,the antisense strand, or both strands. For example, an exemplary siNAmolecule of the invention can comprise about 1 to about 5 or more (e.g.,about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides ornon-nucleotides of Formula II at the 5′-end of the sense strand, theantisense strand, or both strands. In anther non-limiting example, anexemplary siNA molecule of the invention can comprise about 1 to about 5or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modifiednucleotides or non-nucleotides of Formula II at the 3′-end of the sensestrand, the antisense strand, or both strands.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against SCD inside a cell or reconstituted in vitrosystem, wherein the chemical modification comprises one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides ornon-nucleotides having Formula III:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalklylamino, substituted silyl, or group having Formula I or II; R9is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, or any other non-naturallyoccurring base that can be employed to be complementary ornon-complementary to target RNA or a non-nucleosidic base such asphenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone,pyridinone, or any other non-naturally occurring universal base that canbe complementary or non-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Formula III canbe present in one or both oligonucleotide strands of the siNA duplex,for example, in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or morechemically-modified nucleotides or non-nucleotides of Formula III at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand,the antisense strand, or both strands. For example, an exemplary siNAmolecule of the invention can comprise about 1 to about 5 or more (e.g.,about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide(s) ornon-nucleotide(s) of Formula III at the 5′-end of the sense strand, theantisense strand, or both strands. In anther non-limiting example, anexemplary siNA molecule of the invention can comprise about 1 to about 5or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modifiednucleotide or non-nucleotide of Formula III at the 3′-end of the sensestrand, the antisense strand, or both strands.

In another embodiment, a siNA molecule of the invention comprises anucleotide having Formula II or III, wherein the nucleotide havingFormula II or III is in an inverted configuration. For example, thenucleotide having Formula II or III is connected to the siNA constructin a 3′-3′, 3′-2′, 2′-3′, or 5′-5′ configuration, such as at the 3′-end,the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against SCD inside a cell or reconstituted in vitrosystem, wherein the chemical modification comprises a 5′-terminalphosphate group having Formula IV:

wherein each X and Y is independently O, S, N, alkyl, substituted alkyl,or alkylhalo; wherein each Z and W is independently O, S, N, alkyl,substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, alkylhalo, oracetyl; and wherein W, X, Y and Z are not all O.

In one embodiment, the invention features a siNA molecule having a5′-terminal phosphate group having Formula IV on thetarget-complementary strand, for example, a strand complementary to atarget RNA, wherein the siNA molecule comprises an all RNA siNAmolecule. In another embodiment, the invention features a siNA moleculehaving a 5′-terminal phosphate group having Formula IV on thetarget-complementary strand wherein the siNA molecule also comprisesabout 1 to about 3 (e.g., about 1, 2, or 3) nucleotide 3′-terminalnucleotide overhangs having about 1 to about 4 (e.g., about 1, 2, 3, or4) deoxyribonucleotides on the 3′-end of one or both strands. In anotherembodiment, a 5′-terminal phosphate group having Formula IV is presenton the target-complementary strand of a siNA molecule of the invention,for example a siNA molecule having chemical modifications having any ofFormulae I-VII.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against SCD inside a cell or reconstituted in vitrosystem, wherein the chemical modification comprises one or morephosphorothioate internucleotide linkages. For example, in anon-limiting example, the invention features a chemically-modified shortinterfering nucleic acid (siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 ormore phosphorothioate internucleotide linkages in one siNA strand. Inyet another embodiment, the invention features a chemically-modifiedshort interfering nucleic acid (siNA) individually having about 1, 2, 3,4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in bothsiNA strands. The phosphorothioate internucleotide linkages can bepresent in one or both oligonucleotide strands of the siNA duplex, forexample in the sense strand, the antisense strand, or both strands. ThesiNA molecules of the invention can comprise one or morephosphorothioate internucleotide linkages at the 3′-end, the 5′-end, orboth of the 3′- and 5′-ends of the sense strand, the antisense strand,or both strands. For example, an exemplary siNA molecule of theinvention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3,4, 5, or more) consecutive phosphorothioate internucleotide linkages atthe 5′-end of the sense strand, the antisense strand, or both strands.In another non-limiting example, an exemplary siNA molecule of theinvention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) pyrimidine phosphorothioate internucleotide linkages inthe sense strand, the antisense strand, or both strands. In yet anothernon-limiting example, an exemplary siNA molecule of the invention cancomprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) purine phosphorothioate internucleotide linkages in the sensestrand, the antisense strand, or both strands.

In one embodiment, the invention features a siNA molecule, wherein thesense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or about one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and wherein the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidinenucleotides of the sense and/or antisense siNA strand arechemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without one or more, for example about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′- and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features a siNA molecule, whereinthe sense strand comprises about I to about 5, specifically about 1, 2,3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more(e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, ormore) universal base modified nucleotides, and optionally a terminal capmolecule at the 3-end, the 5′-end, or both of the 3′- and 5′-ends of thesense strand; and wherein the antisense strand comprises about 1 toabout 5 or more, specifically about 1, 2, 3, 4, 5, or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidinenucleotides of the sense and/or antisense siNA strand arechemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without about 1 to about 5 or more, for exampleabout 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features a siNA molecule, wherein theantisense strand comprises one or more, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages,and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and wherein the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidinenucleotides of the sense and/or antisense siNA strand arechemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without one or more, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′ and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features a siNA molecule, whereinthe antisense strand comprises about 1 to about 5 or more, specificallyabout 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the sense strand; and whereinthe antisense strand comprises about 1 to about 5 or more, specificallyabout 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the antisense strand. Inanother embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisensesiNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5, forexample about 1, 2, 3, 4, 5 or more phosphorothioate internucleotidelinkages and/or a terminal cap molecule at the 3′-end, the 5′-end, orboth of the 3′- and 5′-ends, being present in the same or differentstrand.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule having about 1 to about 5 ormore (specifically about 1, 2, 3, 4, 5 or more) phosphorothioateinternucleotide linkages in each strand of the siNA molecule.

In another embodiment, the invention features a siNA molecule comprising2′-5′ internucleotide linkages. The 2′-5′ internucleotide linkage(s) canbe at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of one orboth siNA sequence strands. In addition, the 2′-5′ internucleotidelinkage(s) can be present at various other positions within one or bothsiNA sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more including every internucleotide linkage of a pyrimidinenucleotide in one or both strands of the siNA molecule can comprise a2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more including every internucleotide linkage of a purine nucleotidein one or both strands of the siNA molecule can comprise a 2′-5′internucleotide linkage.

In another embodiment, a chemically-modified siNA molecule of theinvention comprises a duplex having two strands, one or both of whichcan be chemically-modified, wherein each strand is independently about15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30) nucleotides in length, wherein the duplex hasabout 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the chemicalmodification comprises a structure having any of Formulae I-VII. Forexample, an exemplary chemically-modified siNA molecule of the inventioncomprises a duplex having two strands, one or both of which can bechemically-modified with a chemical modification having any of FormulaeI-VII or any combination thereof, wherein each strand consists of about21 nucleotides, each having a 2-nucleotide 3 ′-terminal nucleotideoverhang, and wherein the duplex has about 19 base pairs. In anotherembodiment, a siNA molecule of the invention comprises a single strandedhairpin structure, wherein the siNA is about 36 to about 70 (e.g., about36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 15to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30) base pairs, and wherein the siNA can include achemical modification comprising a structure having any of FormulaeI-VII or any combination thereof. For example, an exemplarychemically-modified siNA molecule of the invention comprises a linearoligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45,46, 47, 48, 49, or 50) nucleotides that is chemically-modified with achemical modification having any of Formulae I-VII or any combinationthereof, wherein the linear oligonucleotide forms a hairpin structurehaving about 19 to about 21 (e.g., 19, 20, or 21) base pairs and a2-nucleotide 3′-terminal nucleotide overhang. In another embodiment, alinear hairpin siNA molecule of the invention contains a stem loopmotif, wherein the loop portion of the siNA molecule is biodegradable.For example, a linear hairpin siNA molecule of the invention is designedsuch that degradation of the loop portion of the siNA molecule in vivocan generate a double-stranded siNA molecule with 3′-terminal overhangs,such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

In another embodiment, a siNA molecule of the invention comprises ahairpin structure, wherein the siNA is about 25 to about 50 (e.g., about25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein thesiNA can include one or more chemical modifications comprising astructure having any of Formulae I-VII or any combination thereof. Forexample, an exemplary chemically-modified siNA molecule of the inventioncomprises a linear oligonucleotide having about 25 to about 35 (e.g.,about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that ischemically-modified with one or more chemical modifications having anyof Formulae I-VII or any combination thereof, wherein the linearoligonucleotide forms a hairpin structure having about 3 to about 25(e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25) base pairs and a 5′-terminal phosphategroup that can be chemically modified as described herein (for example a5′-terminal phosphate group having Formula IV). In another embodiment, alinear hairpin siNA molecule of the invention contains a stem loopmotif, wherein the loop portion of the siNA molecule is biodegradable.In one embodiment, a linear hairpin siNA molecule of the inventioncomprises a loop portion comprising a non-nucleotide linker.

In another embodiment, a siNA molecule of the invention comprises anasymmetric hairpin structure, wherein the siNA is about 25 to about 50(e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in lengthhaving about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, andwherein the siNA can include one or more chemical modificationscomprising a structure having any of Formulae I-VII or any combinationthereof. For example, an exemplary chemically-modified siNA molecule ofthe invention comprises a linear oligonucleotide having about 25 toabout 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35)nucleotides that is chemically-modified with one or more chemicalmodifications having any of Formulae I-VII or any combination thereof,wherein the linear oligonucleotide forms an asymmetric hairpin structurehaving about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs and a5′-terminal phosphate group that can be chemically modified as describedherein (for example a 5′-terminal phosphate group having Formula IV). Inone embodiment, an asymmetric hairpin siNA molecule of the inventioncontains a stem loop motif, wherein the loop portion of the siNAmolecule is biodegradable. In another embodiment, an asymmetric hairpinsiNA molecule of the invention comprises a loop portion comprising anon-nucleotide linker.

In another embodiment, a siNA molecule of the invention comprises anasymmetric double stranded structure having separate polynucleotidestrands comprising sense and antisense regions, wherein the antisenseregion is about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length, whereinthe sense region is about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25)nucleotides in length, wherein the sense region and the antisense regionhave at least 3 complementary nucleotides, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. For example, anexemplary chemically-modified siNA molecule of the invention comprisesan asymmetric double stranded structure having separate polynucleotidestrands comprising sense and antisense regions, wherein the antisenseregion is about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23)nucleotides in length and wherein the sense region is about 3 to about15 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15)nucleotides in length, wherein the sense region the antisense regionhave at least 3 complementary nucleotides, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. In another embodiment,the asymmetric double stranded siNA molecule can also have a 5′-terminalphosphate group that can be chemically modified as described herein (forexample a 5′-terminal phosphate group having Formula IV).

In another embodiment, a siNA molecule of the invention comprises acircular nucleic acid molecule, wherein the siNA is about 38 to about 70(e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in lengthhaving about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the siNA caninclude a chemical modification, which comprises a structure having anyof Formulae I-VII or any combination thereof. For example, an exemplarychemically-modified siNA molecule of the invention comprises a circularoligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45,46, 47, 48, 49, or 50) nucleotides that is chemically-modified with achemical modification having any of Formulae I-VII or any combinationthereof, wherein the circular oligonucleotide forms a dumbbell shapedstructure having about 19 base pairs and 2 loops.

In another embodiment, a circular siNA molecule of the inventioncontains two loop motifs, wherein one or both loop portions of the siNAmolecule is biodegradable. For example, a circular siNA molecule of theinvention is designed such that degradation of the loop portions of thesiNA molecule in vivo can generate a double-stranded siNA molecule with3′-terminal overhangs, such as 3′-terminal nucleotide overhangscomprising about 2 nucleotides.

In one embodiment, a siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety,for example a compound having Formula V:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalklylamino, substituted silyl, or group havingFormula I or II; R9 is O, S, CH2, S═O, CHF, or CF2.

In one embodiment, a siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasicmoiety, for example a compound having Formula VI:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalklylamino, substituted silyl, or group havingFormula I or II; R9 is O, S, CH2, S═O, CHF, or CF2, and either R2, R3,R8 or R13 serve as points of attachment to the siNA molecule of theinvention.

In another embodiment, a siNA molecule of the invention comprises atleast one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)substituted polyalkyl moieties, for example a compound having FormulaVII:

wherein each n is independently an integer from 1 to 12, each R1, R2 andR3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl,F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, or a group havingFormula I, and R1, R2 or R3 serves as points of attachment to the siNAmolecule of the invention.

In another embodiment, the invention features a compound having FormulaVII, wherein R1 and R2 are hydroxyl (OH) groups, n=1, and R3 comprises Oand is the point of attachment to the 3′-end, the 5′-end, or both of the3′ and 5′-ends of one or both strands of a double-stranded siNA moleculeof the invention or to a single-stranded siNA molecule of the invention.This modification is referred to herein as “glyceryl” (for examplemodification 6 in FIG. 10).

In another embodiment, a chemically modified nucleoside ornon-nucleoside (e.g. a moiety having any of Formula V, VI or VII) of theinvention is at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends ofa siNA molecule of the invention. For example, chemically modifiednucleoside or non-nucleoside (e.g., a moiety having Formula V, VI orVII) can be present at the 3′-end, the 5′-end, or both of the 3′ and5′-ends of the antisense strand, the sense strand, or both antisense andsense strands of the siNA molecule. In one embodiment, the chemicallymodified nucleoside or non-nucleoside (e.g., a moiety having Formula V,VI or VII) is present at the 5′-end and 3′-end of the sense strand andthe 3′-end of the antisense strand of a double stranded siNA molecule ofthe invention. In one embodiment, the chemically modified nucleoside ornon-nucleoside (e.g., a moiety having Formula V, VI or VII) is presentat the terminal position of the 5′-end and 3′-end of the sense strandand the 3′-end of the antisense strand of a double stranded siNAmolecule of the invention. In one embodiment, the chemically modifiednucleoside or non-nucleoside (e.g., a moiety having Formula V, VI orVII) is present at the two terminal positions of the 5′-end and 3′-endof the sense strand and the 3′-end of the antisense strand of a doublestranded siNA molecule of the invention. In one embodiment, thechemically modified nucleoside or non-nucleoside (e.g., a moiety havingFormula V, VI or VII) is present at the penultimate position of the5′-end and 3′-end of the sense strand and the 3′-end of the antisensestrand of a double stranded siNA molecule of the invention. In addition,a moiety having Formula VII can be present at the 3′-end or the 5′-endof a hairpin siNA molecule as described herein.

In another embodiment, a siNA molecule of the invention comprises anabasic residue having Formula V or VI, wherein the abasic residue havingFormula VI or VI is connected to the siNA construct in a 3′-3′, 3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, a siNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleicacid (LNA) nucleotides, for example, at the 5′-end, the 3′-end, both ofthe 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In another embodiment, a siNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclicnucleotides, for example, at the 5′-end, the 3′-end, both of the 5′ and3′-ends, or any combination thereof, of the siNA molecule.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides),wherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),wherein any (e.g., one or more or all) purine nucleotides present in thesense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), andwherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-O-methyl purine nucleotides (e.g., whereinall purine nucleotides are 2′-O-methyl purine nucleotides or alternatelya plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),wherein any (e.g., one or more or all) purine nucleotides present in theantisense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), andwherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said antisense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-O-methyl purine nucleotides (e.g., whereinall purine nucleotides are 2′-O-methyl purine nucleotides or alternatelya plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention capable ofmediating RNA interference (RNAi) against SCD inside a cell orreconstituted in vitro system comprising a sense region, wherein one ormore pyrimidine nucleotides present in the sense region are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and one or more purine nucleotides present in the senseregion are 2′-deoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-deoxy purine nucleotides or alternately a pluralityof purine nucleotides are 2′-deoxy purine nucleotides), and an antisenseregion, wherein one or more pyrimidine nucleotides present in theantisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g.,wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides or alternately a plurality of pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides), and one or more purinenucleotides present in the antisense region are 2′-O-methyl purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides). The sense region and/or the antisenseregion can have a terminal cap modification, such as any modificationdescribed herein or shown in FIG. 10, that is optionally present at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense and/orantisense sequence. The sense and/or antisense region can optionallyfurther comprise a 3′-terminal nucleotide overhang having about 1 toabout 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides. The overhangnucleotides can further comprise one or more (e.g., about 1, 2, 3, 4 ormore) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetateinternucleotide linkages. Non-limiting examples of thesechemically-modified siNAs are shown in FIGS. 4 and 5 and Tables III andIV herein. In any of these described embodiments, the purine nucleotidespresent in the sense region are alternatively 2′-O-methyl purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides) and one or more purine nucleotidespresent in the antisense region are 2′-O-methyl purine nucleotides(e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotidesor alternately a plurality of purine nucleotides are 2′-O-methyl purinenucleotides). Also, in any of these embodiments, one or more purinenucleotides present in the sense region are alternatively purineribonucleotides (e.g., wherein all purine nucleotides are purineribonucleotides or alternately a plurality of purine nucleotides arepurine ribonucleotides) and any purine nucleotides present in theantisense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides).Additionally, in any of these embodiments, one or more purinenucleotides present in the sense region and/or present in the antisenseregion are alternatively selected from the group consisting of 2′-deoxynucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethylnucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides (e.g.,wherein all purine nucleotides are selected from the group consisting of2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides,2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methylnucleotides or alternately a plurality of purine nucleotides areselected from the group consisting of 2′-deoxy nucleotides, lockednucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides,4′-thionucleotides, and 2′-O-methyl nucleotides).

In another embodiment, any modified nucleotides present in the siNAmolecules of the invention, preferably in the antisense strand of thesiNA molecules of the invention, but also optionally in the sense and/orboth antisense and sense strands, comprise modified nucleotides havingproperties or characteristics similar to naturally occurringribonucleotides. For example, the invention features siNA moleculesincluding modified nucleotides having a Northern conformation (e.g.,Northern pseudorotation cycle, see for example Saenger, Principles ofNucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemicallymodified nucleotides present in the siNA molecules of the invention,preferably in the antisense strand of the siNA molecules of theinvention, but also optionally in the sense and/or both antisense andsense strands, are resistant to nuclease degradation while at the sametime maintaining the capacity to mediate RNAi. Non-limiting examples ofnucleotides having a northern configuration include locked nucleic acid(LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl)nucleotides); 2′-methoxyethoxy (MOE) nucleotides; 2′-methyl-thio-ethyl,2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azidonucleotides, and 2′-O-methyl nucleotides.

In one embodiment, the sense strand of a double stranded siNA moleculeof the invention comprises a terminal cap moiety, (see for example FIG.10) such as an inverted deoxyabaisc moiety, at the 3′-end, 5′-end, orboth 3′ and 5′-ends of the sense strand.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid molecule (siNA) capable of mediating RNAinterference (RNAi) against SCD inside a cell or reconstituted in vitrosystem, wherein the chemical modification comprises a conjugatecovalently attached to the chemically-modified siNA molecule.Non-limiting examples of conjugates contemplated by the inventioninclude conjugates and ligands described in Vargeese et al., U.S. Ser.No. 10/427,160, filed Apr. 30, 2003, incorporated by reference herein inits entirety, including the drawings. In another embodiment, theconjugate is covalently attached to the chemically-modified siNAmolecule via a biodegradable linker. In one embodiment, the conjugatemolecule is attached at the 3′-end of either the sense strand, theantisense strand, or both strands of the chemically-modified siNAmolecule. In another embodiment, the conjugate molecule is attached atthe 5′-end of either the sense strand, the antisense strand, or bothstrands of the chemically-modified siNA molecule. In yet anotherembodiment, the conjugate molecule is attached both the 3′-end and5′-end of either the sense strand, the antisense strand, or both strandsof the chemically-modified siNA molecule, or any combination thereof. Inone embodiment, a conjugate molecule of the invention comprises amolecule that facilitates delivery of a chemically-modified siNAmolecule into a biological system, such as a cell. In anotherembodiment, the conjugate molecule attached to the chemically-modifiedsiNA molecule is a polyethylene glycol, human serum albumin, or a ligandfor a cellular receptor that can mediate cellular uptake. Examples ofspecific conjugate molecules contemplated by the instant invention thatcan be attached to chemically-modified siNA molecules are described inVargeese et al., U.S. Ser. No. 10/201,394, filed Jul. 22, 2002incorporated by reference herein. The type of conjugates used and theextent of conjugation of siNA molecules of the invention can beevaluated for improved pharmacokinetic profiles, bioavailability, and/orstability of siNA constructs while at the same time maintaining theability of the siNA to mediate RNAi activity. As such, one skilled inthe art can screen siNA constructs that are modified with variousconjugates to determine whether the siNA conjugate complex possessesimproved properties while maintaining the ability to mediate RNAi, forexample in animal models as are generally known in the art.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule of the invention, wherein the siNA furthercomprises a nucleotide, non-nucleotide, or mixednucleotide/non-nucleotide linker that joins the sense region of the siNAto the antisense region of the siNA. In one embodiment, a nucleotidelinker of the invention can be a linker of ≧2 nucleotides in length, forexample about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Inanother embodiment, the nucleotide linker can be a nucleic acid aptamer.By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleicacid molecule that binds specifically to a target molecule wherein thenucleic acid molecule has sequence that comprises a sequence recognizedby the target molecule in its natural setting. Alternately, an aptamercan be a nucleic acid molecule that binds to a target molecule where thetarget molecule does not naturally bind to a nucleic acid. The targetmolecule can be any molecule of interest. For example, the aptamer canbe used to bind to a ligand-binding domain of a protein, therebypreventing interaction of the naturally occurring ligand with theprotein. This is a non-limiting example and those in the art willrecognize that other embodiments can be readily generated usingtechniques generally known in the art. (See, for example, Gold et al.,1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J.Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser,2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287,820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.)

In yet another embodiment, a non-nucleotide linker of the inventioncomprises abasic nucleotide, polyether, polyamine, polyamide, peptide,carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g.polyethylene glycols such as those having between 2 and 100 ethyleneglycol units). Specific examples include those described by Seela andKaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987,15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324;Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al.,Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751; Durandet al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides &Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301;Ono et al., Biochemistry 1991, 30:9914; Arnold et al., InternationalPublication No. WO 89/02439; Usman et al., International Publication No.WO 95/06731; Dudycz et al., International Publication No. WO 95/11910and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all herebyincorporated by reference herein. A “non-nucleotide” further means anygroup or compound that can be incorporated into a nucleic acid chain inthe place of one or more nucleotide units, including either sugar and/orphosphate substitutions, and allows the remaining bases to exhibit theirenzymatic activity. The group or compound can be abasic in that it doesnot contain a commonly recognized nucleotide base, such as adenosine,guanine, cytosine, uracil or thymine, for example at the C l position ofthe sugar.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule capable of mediating RNA interference (RNAi) insidea cell or reconstituted in vitro system, wherein one or both strands ofthe siNA molecule that are assembled from two separate oligonucleotidesdo not comprise any ribonucleotides. For example, a siNA molecule can beassembled from a single oligonculeotide where the sense and antisenseregions of the siNA comprise separate oligonucleotides that do not haveany ribonucleotides (e.g., nucleotides having a 2′-OH group) present inthe oligonucleotides. In another example, a siNA molecule can beassembled from a single oligonculeotide where the sense and antisenseregions of the siNA are linked or circularized by a nucleotide ornon-nucleotide linker as described herein, wherein the oligonucleotidedoes not have any ribonucleotides (e.g., nucleotides having a 2′-OHgroup) present in the oligonucleotide. Applicant has surprisingly foundthat the presense of ribonucleotides (e.g., nucleotides having a2′-hydroxyl group) within the siNA molecule is not required or essentialto support RNAi activity. As such, in one embodiment, all positionswithin the siNA can include chemically modified nucleotides and/ornon-nucleotides such as nucleotides and or non-nucleotides havingFormula I, II, III, IV, V, VI, or VII or any combination thereof to theextent that the ability of the siNA molecule to support RNAi activity ina cell is maintained.

In one embodiment, a siNA molecule of the invention is a single strandedsiNA molecule that mediates RNAi activity in a cell or reconstituted invitro system comprising a single stranded polynucleotide havingcomplementarity to a target nucleic acid sequence. In anotherembodiment, the single stranded siNA molecule of the invention comprisesa 5′-terminal phosphate group. In another embodiment, the singlestranded siNA molecule of the invention comprises a 5′-terminalphosphate group and a 3′-terminal phosphate group (e.g., a 2′,3′-cyclicphosphate). In another embodiment, the single stranded siNA molecule ofthe invention comprises about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. Inyet another embodiment, the single stranded siNA molecule of theinvention comprises one or more chemically modified nucleotides ornon-nucleotides described herein. For example, all the positions withinthe siNA molecule can include chemically-modified nucleotides such asnucleotides having any of Formulae I-VII, or any combination thereof tothe extent that the ability of the siNA molecule to support RNAiactivity in a cell is maintained.

In one embodiment, a siNA molecule of the invention is a single strandedsiNA molecule that mediates RNAi activity in a cell or reconstituted invitro system comprising a single stranded polynucleotide havingcomplementarity to a target nucleic acid sequence, wherein one or morepyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any purine nucleotides present in the antisense region are2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are2′-O-methyl purine nucleotides or alternately a plurality of purinenucleotides are 2′-O-methyl purine nucleotides), and a terminal capmodification, such as any modification described herein or shown in FIG.10, that is optionally present at the 3′-end, the 5′-end, or both of the3′ and 5′-ends of the antisense sequence. The siNA optionally furthercomprises about 1 to about 4 or more (e.g., about 1, 2, 3, 4 or more)terminal 2′-deoxynucleotides at the 3′-end of the siNA molecule, whereinthe terminal nucleotides can further comprise one or more (e.g., 1, 2,3, 4 or more) phosphorothioate, phosphonoacetate, and/orthiophosphonoacetate internucleotide linkages, and wherein the siNAoptionally further comprises a terminal phosphate group, such as a5′-terminal phosphate group. In any of these embodiments, any purinenucleotides present in the antisense region are alternatively 2′-deoxypurine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxypurine nucleotides or alternately a plurality of purine nucleotides are2′-deoxy purine nucleotides). Also, in any of these embodiments, anypurine nucleotides present in the siNA (i.e., purine nucleotides presentin the sense and/or antisense region) can alternatively be lockednucleic acid (LNA) nucleotides (e.g., wherein all purine nucleotides areLNA nucleotides or alternately a plurality of purine nucleotides are LNAnucleotides). Also, in any of these embodiments, any purine nucleotidespresent in the siNA are alternatively 2′-methoxyethyl purine nucleotides(e.g., wherein all purine nucleotides are 2′-methoxyethyl purinenucleotides or alternately a plurality of purine nucleotides are2′-methoxyethyl purine nucleotides). In another embodiment, any modifiednucleotides present in the single stranded siNA molecules of theinvention comprise modified nucleotides having properties orcharacteristics similar to naturally occurring ribonucleotides. Forexample, the invention features siNA molecules including modifiednucleotides having a Northern conformation (e.g., Northernpseudorotation cycle, see for example Saenger, Principles of NucleicAcid Structure, Springer-Verlag ed., 1984). As such, chemically modifiednucleotides present in the single stranded siNA molecules of theinvention are preferably resistant to nuclease degradation while at thesame time maintaining the capacity to mediate RNAi.

In one embodiment, a siNA molecule of the invention comprises chemicallymodified nucleotides or non-nucleotides (e.g., having any of FormulaeI-VII, such as 2′-deoxy, 2′-deoxy-2′-fluoro, or 2′-O-methyl nucleotides)at alternating positions within one or more strands or regions of thesiNA molecule. For example, such chemical modifications can beintroduced at every other position of a RNA based siNA molecule,starting at either the first or second nucleotide from the 3′-end or5′-end of the siNA. In a non-limiting example, a double stranded siNAmolecule of the invention in which each strand of the siNA is 21nucleotides in length is featured wherein positions 1, 3, 5, 7, 9, 11,13, 15, 17, 19 and 21 of each strand are chemically modified (e.g., withcompounds having any of Formulae l-VII, such as such as 2′-deoxy,2′-deoxy-2′-fluoro, or 2′-O-methyl nucleotides). In another non-limitingexample, a double stranded siNA molecule of the invention in which eachstrand of the siNA is 21 nucleotides in length is featured whereinpositions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 of each strand arechemically modified (e.g., with compounds having any of Formulae I-VII,such as such as 2′-deoxy, 2′-deoxy-2′-fluoro, or 2′-O-methylnucleotides). Such siNA molecules can further comprise terminal capmoieties and/or backbone modifications as described herein.

In one embodiment, the invention features a method for modulating theexpression of a SCD gene within a cell comprising: (a) synthesizing asiNA molecule of the invention, which can be chemically-modified,wherein one of the siNA strands comprises a sequence complementary toRNA of the SCD gene; and (b) introducing the siNA molecule into a cellunder conditions suitable to modulate the expression of the SCD gene inthe cell.

In one embodiment, the invention features a method for modulating theexpression of a SCD gene within a cell comprising: (a) synthesizing asiNA molecule of the invention, which can be chemically-modified,wherein one of the siNA strands comprises a sequence complementary toRNA of the SCD gene and wherein the sense strand sequence of the siNAcomprises a sequence identical or substantially similar to the sequenceof the target RNA; and (b) introducing the siNA molecule into a cellunder conditions suitable to modulate the expression of the SCD gene inthe cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one SCD gene within a cell comprising: (a)synthesizing siNA molecules of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the SCD genes; and (b) introducing thesiNA molecules into a cell under conditions suitable to modulate theexpression of the SCD genes in the cell.

In another embodiment, the invention features a method for modulatingthe expression of two or more SCD genes within a cell comprising: (a)synthesizing one or more siNA molecules of the invention, which can bechemically-modified, wherein the siNA strands comprise sequencescomplementary to RNA of the SCD genes and wherein the sense strandsequences of the siNAs comprise sequences identical or substantiallysimilar to the sequences of the target RNAs; and (b) introducing thesiNA molecules into a cell under conditions suitable to modulate theexpression of the SCD genes in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one SCD gene within a cell comprising: (a)synthesizing a siNA molecule of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the SCD gene and wherein the sensestrand sequence of the siNA comprises a sequence identical orsubstantially similar to the sequences of the target RNAs; and (b)introducing the siNA molecule into a cell under conditions suitable tomodulate the expression of the SCD genes in the cell.

In one embodiment, siNA molecules of the invention are used as reagentsin ex vivo applications. For example, siNA reagents are introduced intotissue or cells that are transplanted into a subject for therapeuticeffect. The cells and/or tissue can be derived from an organism orsubject that later receives the explant, or can be derived from anotherorganism or subject prior to transplantation. The siNA molecules can beused to modulate the expression of one or more genes in the cells ortissue, such that the cells or tissue obtain a desired phenotype or areable to perform a function when transplanted in vivo. In one embodiment,certain target cells from a patient are extracted. These extracted cellsare contacted with siNAs targeting a specific nucleotide sequence withinthe cells under conditions suitable for uptake of the siNAs by thesecells (e.g. using delivery reagents such as cationic lipids, liposomesand the like or using techniques such as electroporation to facilitatethe delivery of siNAs into cells). The cells are then reintroduced backinto the same patient or other patients. In one embodiment, theinvention features a method of modulating the expression of a SCD genein a tissue explant comprising: (a) synthesizing a siNA molecule of theinvention, which can be chemically-modified, wherein one of the siNAstrands comprises a sequence complementary to RNA of the SCD gene; and(b) introducing the siNA molecule into a cell of the tissue explantderived from a particular organism under conditions suitable to modulatethe expression of the SCD gene in the tissue explant. In anotherembodiment, the method further comprises introducing the tissue explantback into the organism the tissue was derived from or into anotherorganism under conditions suitable to modulate the expression of the SCDgene in that organism.

In one embodiment, the invention features a method of modulating theexpression of a SCD gene in a tissue explant comprising: (a)synthesizing a siNA molecule of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the SCD gene and wherein the sensestrand sequence of the siNA comprises a sequence identical orsubstantially similar to the sequence of the target RNA; and (b)introducing the siNA molecule into a cell of the tissue explant derivedfrom a particular organism under conditions suitable to modulate theexpression of the SCD gene in the tissue explant. In another embodiment,the method further comprises introducing the tissue explant back intothe organism the tissue was derived from or into another organism underconditions suitable to modulate the expression of the SCD gene in thatorganism.

In another embodiment, the invention features a method of modulating theexpression of more than one SCD gene in a tissue explant comprising: (a)synthesizing siNA molecules of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the SCD genes; and (b) introducing thesiNA molecules into a cell of the tissue explant derived from aparticular organism under conditions suitable to modulate the expressionof the SCD genes in the tissue explant. In another embodiment, themethod further comprises introducing the tissue explant back into theorganism the tissue was derived from or into another organism underconditions suitable to modulate the expression of the SCD genes in thatorganism.

In one embodiment, the invention features a method of modulating theexpression of a SCD gene in a subject or organism comprising: (a)synthesizing a siNA molecule of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the SCD gene; and (b) introducing thesiNA molecule into the subject or organism under conditions suitable tomodulate the expression of the SCD gene in the subject or organism. Thelevel of SCD protein or RNA can be determined using various methodswell-known in the art.

In another embodiment, the invention features a method of modulating theexpression of more than one SCD gene in a subject or organismcomprising: (a) synthesizing siNA molecules of the invention, which canbe chemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the SCD genes; and (b) introducing thesiNA molecules into the subject or organism under conditions suitable tomodulate the expression of the SCD genes in the subject or organism. Thelevel of SCD protein or RNA can be determined as is known in the art.

In one embodiment, the invention features a method for modulating theexpression of a SCD gene within a cell comprising: (a) synthesizing asiNA molecule of the invention, which can be chemically-modified,wherein the siNA comprises a single stranded sequence havingcomplementarity to RNA of the SCD gene; and (b) introducing the siNAmolecule into a cell under conditions suitable to modulate theexpression of the SCD gene in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one SCD gene within a cell comprising: (a)synthesizing siNA molecules of the invention, which can bechemically-modified, wherein the siNA comprises a single strandedsequence having complementarity to RNA of the SCD gene; and (b)contacting the cell in vitro or in vivo with the siNA molecule underconditions suitable to modulate the expression of the SCD genes in thecell.

In one embodiment, the invention features a method of modulating theexpression of a SCD gene in a tissue explant comprising: (a)synthesizing a siNA molecule of the invention, which can bechemically-modified, wherein the siNA comprises a single strandedsequence having complementarity to RNA of the SCD gene; and (b)contacting a cell of the tissue explant derived from a particularsubject or organism with the siNA molecule under conditions suitable tomodulate the expression of the SCD gene in the tissue explant. Inanother embodiment, the method further comprises introducing the tissueexplant back into the subject or organism the tissue was derived from orinto another subject or organism under conditions suitable to modulatethe expression of the SCD gene in that subject or organism.

In another embodiment, the invention features a method of modulating theexpression of more than one SCD gene in a tissue explant comprising: (a)synthesizing siNA molecules of the invention, which can bechemically-modified, wherein the siNA comprises a single strandedsequence having complementarity to RNA of the SCD gene; and (b)introducing the siNA molecules into a cell of the tissue explant derivedfrom a particular subject or organism under conditions suitable tomodulate the expression of the SCD genes in the tissue explant. Inanother embodiment, the method further comprises introducing the tissueexplant back into the subject or organism the tissue was derived from orinto another subject or organism under conditions suitable to modulatethe expression of the SCD genes in that subject or organism.

In one embodiment, the invention features a method of modulating theexpression of a SCD gene in a subject or organism comprising: (a)synthesizing a siNA molecule of the invention, which can bechemically-modified, wherein the siNA comprises a single strandedsequence having complementarity to RNA of the SCD gene; and (b)introducing the siNA molecule into the subject or organism underconditions suitable to modulate the expression of the SCD gene in thesubject or organism.

In another embodiment, the invention features a method of modulating theexpression of more than one SCD gene in a subject or organismcomprising: (a) synthesizing siNA molecules of the invention, which canbe chemically-modified, wherein the siNA comprises a single strandedsequence having complementarity to RNA of the SCD gene; and (b)introducing the siNA molecules into the subject or organism underconditions suitable to modulate the expression of the SCD genes in thesubject or organism.

In one embodiment, the invention features a method of modulating theexpression of a SCD gene in a subject or organism comprising contactingthe subject or organism with a siNA molecule of the invention underconditions suitable to modulate the expression of the SCD gene in thesubject or organism.

In one embodiment, the invention features a method for treating orpreventing diabetes (type I and/or type II) in a subject or organismcomprising contacting the subject or organism with a siNA molecule ofthe invention under conditions suitable to modulate the expression ofthe SCD gene in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing atherosclerosis in a subject or organism comprisingcontacting the subject or organism with a siNA molecule of the inventionunder conditions suitable to modulate the expression of the SCD gene inthe subject or organism.

In one embodiment, the invention features a method for treating orpreventing cancer in a subject or organism comprising contacting thesubject or organism with a siNA molecule of the invention underconditions suitable to modulate the expression of the SCD gene in thesubject or organism.

In one embodiment, the invention features a method for treating orpreventing obesity in a subject or organism comprising contacting thesubject or organism with a siNA molecule of the invention underconditions suitable to modulate the expression of the SCD gene in thesubject or organism.

In one embodiment, the invention features a method for inducing weightloss in a subject or organism comprising contacting the subject ororganism with a siNA molecule of the invention under conditions suitableto modulate the expression of the SCD gene in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing a viral infection in a subject or organism comprisingcontacting the subject or organism with a siNA molecule of the inventionunder conditions suitable to modulate the expression of the SCD gene inthe subject or organism.

In another embodiment, the invention features a method of modulating theexpression of more than one SCD genes in a subject or organismcomprising contacting the subject or organism with one or more siNAmolecules of the invention under conditions suitable to modulate theexpression of the SCD genes in the subject or organism.

The siNA molecules of the invention can be designed to down regulate orinhibit target (e.g., SCD) gene expression through RNAi targeting of avariety of RNA molecules. In one embodiment, the siNA molecules of theinvention are used to target various RNAs corresponding to a targetgene. Non-limiting examples of such RNAs include messenger RNA (mRNA),alternate RNA splice variants of target gene(s), post-transcriptionallymodified RNA of target gene(s), pre-mRNA of target gene(s), and/or RNAtemplates. If alternate splicing produces a family of transcripts thatare distinguished by usage of appropriate exons, the instant inventioncan be used to inhibit gene expression through the appropriate exons tospecifically inhibit or to distinguish among the functions of genefamily members. For example, a protein that contains an alternativelyspliced transmembrane domain can be expressed in both membrane bound andsecreted forms. Use of the invention to target the exon containing thetransmembrane domain can be used to determine the functionalconsequences of pharmaceutical targeting of membrane bound as opposed tothe secreted form of the protein. Non-limiting examples of applicationsof the invention relating to targeting these RNA molecules includetherapeutic pharmaceutical applications, pharmaceutical discoveryapplications, molecular diagnostic and gene function applications, andgene mapping, for example using single nucleotide polymorphism mappingwith siNA molecules of the invention. Such applications can beimplemented using known gene sequences or from partial sequencesavailable from an expressed sequence tag (EST).

In another embodiment, the siNA molecules of the invention are used totarget conserved sequences corresponding to a gene family or genefamilies such as SCD family genes. As such, siNA molecules targetingmultiple SCD targets can provide increased therapeutic effect. Inaddition, siNA can be used to characterize pathways of gene function ina variety of applications. For example, the present invention can beused to inhibit the activity of target gene(s) in a pathway to determinethe function of uncharacterized gene(s) in gene function analysis, mRNAfunction analysis, or translational analysis. The invention can be usedto determine potential target gene pathways involved in various diseasesand conditions toward pharmaceutical development. The invention can beused to understand pathways of gene expression involved in, for examplediabetes (type I and/or type II), atherosclerosis, cancer, obesity, andviral infection.

In one embodiment, siNA molecule(s) and/or methods of the invention areused to down regulate the expression of gene(s) that encode RNA referredto by Genbank Accession, for example, SCD genes encoding RNA sequence(s)referred to herein by Genbank Accession number, for example, GenbankAccession Nos. shown in Table I.

In one embodiment, the invention features a method comprising: (a)generating a library of siNA constructs having a predeterminedcomplexity; and (b) assaying the siNA constructs of (a) above, underconditions suitable to determine RNAi target sites within the target RNAsequence. In one embodiment, the siNA molecules of (a) have strands of afixed length, for example, about 23 nucleotides in length. In anotherembodiment, the siNA molecules of (a) are of differing length, forexample having strands of about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides inlength. In one embodiment, the assay can comprise a reconstituted invitro siNA assay as described herein. In another embodiment, the assaycan comprise a cell culture system in which target RNA is expressed. Inanother embodiment, fragments of target RNA are analyzed for detectablelevels of cleavage, for example by gel electrophoresis, northern blotanalysis, or RNAse protection assays, to determine the most suitabletarget site(s) within the target RNA sequence. The target RNA sequencecan be obtained as is known in the art, for example, by cloning and/ortranscription for in vitro systems, and by cellular expression in invivo systems.

In one embodiment, the invention features a method comprising: (a)generating a randomized library of siNA constructs having apredetermined complexity, such as of 4N, where N represents the numberof base paired nucleotides in each of the siNA construct strands (eg.for a siNA construct having 21 nucleotide sense and antisense strandswith 19 base pairs, the complexity would be 419); and (b) assaying thesiNA constructs of (a) above, under conditions suitable to determineRNAi target sites within the target SCD RNA sequence. In anotherembodiment, the siNA molecules of (a) have strands of a fixed length,for example about 23 nucleotides in length. In yet another embodiment,the siNA molecules of (a) are of differing length, for example havingstrands of about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length. In oneembodiment, the assay can comprise a reconstituted in vitro siNA assayas described in Example 6 herein. In another embodiment, the assay cancomprise a cell culture system in which target RNA is expressed. Inanother embodiment, fragments of SCD RNA are analyzed for detectablelevels of cleavage, for example, by gel electrophoresis, northern blotanalysis, or RNAse protection assays, to determine the most suitabletarget site(s) within the target SCD RNA sequence. The target SCD RNAsequence can be obtained as is known in the art, for example, by cloningand/or transcription for in vitro systems, and by cellular expression inin vivo systems.

In another embodiment, the invention features a method comprising: (a)analyzing the sequence of a RNA target encoded by a target gene; (b)synthesizing one or more sets of siNA molecules having sequencecomplementary to one or more regions of the RNA of (a); and (c) assayingthe siNA molecules of (b) under conditions suitable to determine RNAitargets within the target RNA sequence. In one embodiment, the siNAmolecules of (b) have strands of a fixed length, for example about 23nucleotides in length. In another embodiment, the siNA molecules of (b)are of differing length, for example having strands of about 15 to about30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30) nucleotides in length. In one embodiment, the assay cancomprise a reconstituted in vitro siNA assay as described herein. Inanother embodiment, the assay can comprise a cell culture system inwhich target RNA is expressed. Fragments of target RNA are analyzed fordetectable levels of cleavage, for example by gel electrophoresis,northern blot analysis, or RNAse protection assays, to determine themost suitable target site(s) within the target RNA sequence. The targetRNA sequence can be obtained as is known in the art, for example, bycloning and/or transcription for in vitro systems, and by expression inin vivo systems.

By “target site” is meant a sequence within a target RNA that is“targeted” for cleavage mediated by a siNA construct which containssequences within its antisense region that are complementary to thetarget sequence.

By “detectable level of cleavage” is meant cleavage of target RNA (andformation of cleaved product RNAs) to an extent sufficient to discerncleavage products above the background of RNAs produced by randomdegradation of the target RNA. Production of cleavage products from 1-5%of the target RNA is sufficient to detect above the background for mostmethods of detection.

In one embodiment, the invention features a composition comprising asiNA molecule of the invention, which can be chemically-modified, in apharmaceutically acceptable carrier or diluent. In another embodiment,the invention features a pharmaceutical composition comprising siNAmolecules of the invention, which can be chemically-modified, targetingone or more genes in a pharmaceutically acceptable carrier or diluent.In another embodiment, the invention features a method for diagnosing adisease or condition in a subject comprising administering to thesubject a composition of the invention under conditions suitable for thediagnosis of the disease or condition in the subject. In anotherembodiment, the invention features a method for treating or preventing adisease or condition in a subject, comprising administering to thesubject a composition of the invention under conditions suitable for thetreatment or prevention of the disease or condition in the subject,alone or in conjunction with one or more other therapeutic compounds. Inyet another embodiment, the invention features a method for treating orpreventing diabetes (type I and/or type II), atherosclerosis, cancer,obesity, and viral infection in a subject or organism comprisingadministering to the subject a composition of the invention underconditions suitable for the treatment or prevention of diabetes (type Iand/or type II), atherosclerosis, cancer, obesity, and viral infectionin the subject or organism.

In another embodiment, the invention features a method for validating aSCD gene target, comprising: (a) synthesizing a siNA molecule of theinvention, which can be chemically-modified, wherein one of the siNAstrands includes a sequence complementary to RNA of a SCD target gene;(b) introducing the siNA molecule into a cell, tissue, subject, ororganism under conditions suitable for modulating expression of the SCDtarget gene in the cell, tissue, subject, or organism; and (c)determining the function of the gene by assaying for any phenotypicchange in the cell, tissue, subject, or organism.

In another embodiment, the invention features a method for validating aSCD target comprising: (a) synthesizing a siNA molecule of theinvention, which can be chemically-modified, wherein one of the siNAstrands includes a sequence complementary to RNA of a SCD target gene;(b) introducing the siNA molecule into a biological system underconditions suitable for modulating expression of the SCD target gene inthe biological system; and (c) determining the function of the gene byassaying for any phenotypic change in the biological system.

By “biological system” is meant, material, in a purified or unpurifiedform, from biological sources, including but not limited to human oranimal, wherein the system comprises the components required for RNAiactivity. The term “biological system” includes, for example, a cell,tissue, subject, or organism, or extract thereof. The term biologicalsystem also includes reconstituted RNAi systems that can be used in anin vitro setting.

By “phenotypic change” is meant any detectable change to a cell thatoccurs in response to contact or treatment with a nucleic acid moleculeof the invention (e.g., siNA). Such detectable changes include, but arenot limited to, changes in shape, size, proliferation, motility, proteinexpression or RNA expression or other physical or chemical changes ascan be assayed by methods known in the art. The detectable change canalso include expression of reporter genes/molecules such as GreenFlorescent Protein (GFP) or various tags that are used to identify anexpressed protein or any other cellular component that can be assayed.

In one embodiment, the invention features a kit containing a siNAmolecule of the invention, which can be chemically-modified, that can beused to modulate the expression of a SCD target gene in a biologicalsystem, including, for example, in a cell, tissue, subject, or organism.In another embodiment, the invention features a kit containing more thanone siNA molecule of the invention, which can be chemically-modified,that can be used to modulate the expression of more than one SCD targetgene in a biological system, including, for example, in a cell, tissue,subject, or organism.

In one embodiment, the invention features a cell containing one or moresiNA molecules of the invention, which can be chemically-modified. Inanother embodiment, the cell containing a siNA molecule of the inventionis a mammalian cell. In yet another embodiment, the cell containing asiNA molecule of the invention is a human cell.

In one embodiment, the synthesis of a siNA molecule of the invention,which can be chemically-modified, comprises: (a) synthesis of twocomplementary strands of the siNA molecule; (b) annealing the twocomplementary strands together under conditions suitable to obtain adouble-stranded siNA molecule. In another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phaseoligonucleotide synthesis. In yet another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phase tandemoligonucleotide synthesis.

In one embodiment, the invention features a method for synthesizing asiNA duplex molecule comprising: (a) synthesizing a firstoligonucleotide sequence strand of the siNA molecule, wherein the firstoligonucleotide sequence strand comprises a cleavable linker moleculethat can be used as a scaffold for the synthesis of the secondoligonucleotide sequence strand of the siNA; (b) synthesizing the secondoligonucleotide sequence strand of siNA on the scaffold of the firstoligonucleotide sequence strand, wherein the second oligonucleotidesequence strand further comprises a chemical moiety than can be used topurify the siNA duplex; (c) cleaving the linker molecule of (a) underconditions suitable for the two siNA oligonucleotide strands tohybridize and form a stable duplex; and (d) purifying the siNA duplexutilizing the chemical moiety of the second oligonucleotide sequencestrand. In one embodiment, cleavage of the linker molecule in (c) abovetakes place during deprotection of the oligonucleotide, for example,under hydrolysis conditions using an alkylamine base such asmethylamine. In one embodiment, the method of synthesis comprises solidphase synthesis on a solid support such as controlled pore glass (CPG)or polystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity as thesolid support derivatized linker, such that cleavage of the solidsupport derivatized linker and the cleavable linker of (a) takes placeconcomitantly. In another embodiment, the chemical moiety of (b) thatcan be used to isolate the attached oligonucleotide sequence comprises atrityl group, for example a dimethoxytrityl group, which can be employedin a trityl-on synthesis strategy as described herein. In yet anotherembodiment, the chemical moiety, such as a dimethoxytrityl group, isremoved during purification, for example, using acidic conditions.

In a further embodiment, the method for siNA synthesis is a solutionphase synthesis or hybrid phase synthesis wherein both strands of thesiNA duplex are synthesized in tandem using a cleavable linker attachedto the first sequence which acts a scaffold for synthesis of the secondsequence. Cleavage of the linker under conditions suitable forhybridization of the separate siNA sequence strands results in formationof the double-stranded siNA molecule.

In another embodiment, the invention features a method for synthesizinga siNA duplex molecule comprising: (a) synthesizing one oligonucleotidesequence strand of the siNA molecule, wherein the sequence comprises acleavable linker molecule that can be used as a scaffold for thesynthesis of another oligonucleotide sequence; (b) synthesizing a secondoligonucleotide sequence having complementarity to the first sequencestrand on the scaffold of (a), wherein the second sequence comprises theother strand of the double-stranded siNA molecule and wherein the secondsequence further comprises a chemical moiety than can be used to isolatethe attached oligonucleotide sequence; (c) purifying the product of (b)utilizing the chemical moiety of the second oligonucleotide sequencestrand under conditions suitable for isolating the full-length sequencecomprising both siNA oligonucleotide strands connected by the cleavablelinker and under conditions suitable for the two siNA oligonucleotidestrands to hybridize and form a stable duplex. In one embodiment,cleavage of the linker molecule in (c) above takes place duringdeprotection of the oligonucleotide, for example, under hydrolysisconditions. In another embodiment, cleavage of the linker molecule in(c) above takes place after deprotection of the oligonucleotide. Inanother embodiment, the method of synthesis comprises solid phasesynthesis on a solid support such as controlled pore glass (CPG) orpolystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity ordiffering reactivity as the solid support derivatized linker, such thatcleavage of the solid support derivatized linker and the cleavablelinker of (a) takes place either concomitantly or sequentially. In oneembodiment, the chemical moiety of (b) that can be used to isolate theattached oligonucleotide sequence comprises a trityl group, for examplea dimethoxytrityl group.

In another embodiment, the invention features a method for making adouble-stranded siNA molecule in a single synthetic process comprising:(a) synthesizing an oligonucleotide having a first and a secondsequence, wherein the first sequence is complementary to the secondsequence, and the first oligonucleotide sequence is linked to the secondsequence via a cleavable linker, and wherein a terminal 5′-protectinggroup, for example, a 5′-O-dimethoxytrityl group (5′-O-DMT) remains onthe oligonucleotide having the second sequence; (b) deprotecting theoligonucleotide whereby the deprotection results in the cleavage of thelinker joining the two oligonucleotide sequences; and (c) purifying theproduct of (b) under conditions suitable for isolating thedouble-stranded siNA molecule, for example using a trityl-on synthesisstrategy as described herein.

In another embodiment, the method of synthesis of siNA molecules of theinvention comprises the teachings of Scaringe et al., U.S. Pat. Nos.5,889,136; 6,008,400; and 6,111,086, incorporated by reference herein intheir entirety.

In one embodiment, the invention features siNA constructs that mediateRNAi against SCD, wherein the siNA construct comprises one or morechemical modifications, for example, one or more chemical modificationshaving any of Formulae I-VII or any combination thereof that increasesthe nuclease resistance of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules with increased nuclease resistance comprising (a)introducing nucleotides having any of Formula I-VII or any combinationthereof into a siNA molecule, and (b) assaying the siNA molecule of step(a) under conditions suitable for isolating siNA molecules havingincreased nuclease resistance.

In another embodiment, the invention features a method for generatingsiNA molecules with improved toxicologic profiles (e.g., have attenuatedor no immunstimulatory properties) comprising (a) introducingnucleotides having any of Formula I-VII (e.g., siNA motifs referred toin Table IV) or any combination thereof into a siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved toxicologic profiles.

In another embodiment, the invention features a method for generatingsiNA molecules that do not stimulate an interferon response (e.g., nointerferon response or attenuated interferon response) in a cell,subject, or organism, comprising (a) introducing nucleotides having anyof Formula I-VII (e.g., siNA motifs referred to in Table IV) or anycombination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules that do not stimulate an interferon response.

By “improved toxicologic profile”, is meant that the chemically modifiedsiNA construct exhibits decreased toxicity in a cell, subject, ororganism compared to an unmodified siNA or siNA molecule having fewermodifications or modifications that are less effective in impartingimproved toxicology. In a non-limiting example, siNA molecules withimproved toxicologic profiles are associated with a decreased orattenuated immunostimulatory response in a cell, subject, or organismcompared to an unmodified siNA or siNA molecule having fewermodifications or modifications that are less effective in impartingimproved toxicology. In one embodiment, a siNA molecule with an improvedtoxicological profile comprises no ribonucleotides. In one embodiment, asiNA molecule with an improved toxicological profile comprises less than5 ribonucleotides (e.g., 1, 2, 3, or 4 ribonucleotides). In oneembodiment, a siNA molecule with an improved toxicological profilecomprises Stab 7, Stab 8, Stab 11, Stab 12, Stab 13, Stab 16, Stab 17,Stab 18, Stab 19, Stab 20, Stab 23, Stab 24, Stab 25, Stab 26, Stab 27,Stab 28, Stab 29, Stab 30, Stab 31, Stab 32 or any combination thereof(see Table IV). In one embodiment, the level of immunostimulatoryresponse associated with a given siNA molecule can be measured as isknown in the art, for example by determining the level of PKR/interferonresponse, proliferation, B-cell activation, and/or cytokine productionin assays to quantitate the immunostimulatory response of particularsiNA molecules (see, for example, Leifer et al., 2003, J Immunother. 26,313-9; and U.S. Pat. No. 5,968,909, incorporated in its entirety byreference).

In one embodiment, the invention features siNA constructs that mediateRNAi against SCD, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the bindingaffinity between the sense and antisense strands of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the sense andantisense strands of the siNA molecule comprising (a) introducingnucleotides having any of Formula I-VII or any combination thereof intoa siNA molecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having increasedbinding affinity between the sense and antisense strands of the siNAmolecule.

In one embodiment, the invention features siNA constructs that mediateRNAi against SCD, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the bindingaffinity between the antisense strand of the siNA construct and acomplementary target RNA sequence within a cell.

In one embodiment, the invention features siNA constructs that mediateRNAi against SCD, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the bindingaffinity between the antisense strand of the siNA construct and acomplementary target DNA sequence within a cell.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary target RNA sequencecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary target RNA sequence.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary target DNA sequencecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary target DNA sequence.

In one embodiment, the invention features siNA constructs that mediateRNAi against SCD, wherein the siNA construct comprises one or morechemical modifications described herein that modulate the polymeraseactivity of a cellular polymerase capable of generating additionalendogenous siNA molecules having sequence homology to thechemically-modified siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to a chemically-modified siNAmolecule comprising (a) introducing nucleotides having any of FormulaI-VII or any combination thereof into a siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to the chemically-modified siNAmolecule.

In one embodiment, the invention features chemically-modified siNAconstructs that mediate RNAi against SCD in a cell, wherein the chemicalmodifications do not significantly effect the interaction of siNA with atarget RNA molecule, DNA molecule and/or proteins or other factors thatare essential for RNAi in a manner that would decrease the efficacy ofRNAi mediated by such siNA constructs.

In another embodiment, the invention features a method for generatingsiNA molecules with improved RNAi activity against SCD comprising (a)introducing nucleotides having any of Formula I-VII or any combinationthereof into a siNA molecule, and (b) assaying the siNA molecule of step(a) under conditions suitable for isolating siNA molecules havingimproved RNAi activity.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity against SCD targetRNA comprising (a) introducing nucleotides having any of Formula I-VIIor any combination thereof into a siNA molecule, and (b) assaying thesiNA molecule of step (a) under conditions suitable for isolating siNAmolecules having improved RNAi activity against the target RNA.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity against SCD targetDNA comprising (a) introducing nucleotides having any of Formula I-VIIor any combination thereof into a siNA molecule, and (b) assaying thesiNA molecule of step (a) under conditions suitable for isolating siNAmolecules having improved RNAi activity against the target DNA.

In one embodiment, the invention features siNA constructs that mediateRNAi against SCD, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the cellularuptake of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules against SCD with improved cellular uptake comprising (a)introducing nucleotides having any of Formula I-VII or any combinationthereof into a siNA molecule, and (b) assaying the siNA molecule of step(a) under conditions suitable for isolating siNA molecules havingimproved cellular uptake.

In one embodiment, the invention features siNA constructs that mediateRNAi against SCD, wherein the siNA construct comprises one or morechemical modifications described herein that increases thebioavailability of the siNA construct, for example, by attachingpolymeric conjugates such as polyethyleneglycol or equivalent conjugatesthat improve the pharmacokinetics of the siNA construct, or by attachingconjugates that target specific tissue types or cell types in vivo.Non-limiting examples of such conjugates are described in Vargeese etal., U.S. Ser. No. 10/201,394 incorporated by reference herein.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved bioavailability comprising (a)introducing a conjugate into the structure of a siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchconjugates can include ligands for cellular receptors, such as peptidesderived from naturally occurring protein ligands; protein localizationsequences, including cellular ZIP code sequences; antibodies; nucleicacid aptamers; vitamins and other co-factors, such as folate andN-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG);phospholipids; cholesterol; polyamines, such as spermine or spermidine;and others.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence is chemically modified in amanner that it can no longer act as a guide sequence for efficientlymediating RNA interference and/or be recognized by cellular proteinsthat facilitate RNAi.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein the second sequence is designed or modified in amanner that prevents its entry into the RNAi pathway as a guide sequenceor as a sequence that is complementary to a target nucleic acid (e.g.,RNA) sequence. Such design or modifications are expected to enhance theactivity of siNA and/or improve the specificity of siNA molecules of theinvention. These modifications are also expected to minimize anyoff-target effects and/or associated toxicity.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence is incapable of acting as a guidesequence for mediating RNA interference.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence does not have a terminal5′-hydroxyl (5′-OH) or 5′-phosphate group.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence comprises a terminal cap moietyat the 5′-end of said second sequence. In one embodiment, the terminalcap moiety comprises an inverted abasic, inverted deoxy abasic, invertednucleotide moiety, a group shown in FIG. 10, an alkyl or cycloalkylgroup, a heterocycle, or any other group that prevents RNAi activity inwhich the second sequence serves as a guide sequence or template forRNAi.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence comprises a terminal cap moietyat the 5′-end and 3′-end of said second sequence. In one embodiment,each terminal cap moiety individually comprises an inverted abasic,inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG.10, an alkyl or cycloalkyl group, a heterocycle, or any other group thatprevents RNAi activity in which the second sequence serves as a guidesequence or template for RNAi.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved specificity for down regulatingor inhibiting the expression of a target nucleic acid (e.g., a DNA orRNA such as a gene or its corresponding RNA), comprising (a) introducingone or more chemical modifications into the structure of a siNAmolecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having improvedspecificity. In another embodiment, the chemical modification used toimprove specificity comprises terminal cap modifications at the 5′-end,3′-end, or both 5′ and 3′-ends of the siNA molecule. The terminal capmodifications can comprise, for example, structures shown in FIG. 10(e.g. inverted deoxyabasic moieties) or any other chemical modificationthat renders a portion of the siNA molecule (e.g. the sense strand)incapable of mediating RNA interference against an off target nucleicacid sequence. In a non-limiting example, a siNA molecule is designedsuch that only the antisense sequence of the siNA molecule can serve asa guide sequence for RISC mediated degradation of a corresponding targetRNA sequence. This can be accomplished by rendering the sense sequenceof the siNA inactive by introducing chemical modifications to the sensestrand that preclude recognition of the sense strand as a guide sequenceby RNAi machinery. In one embodiment, such chemical modificationscomprise any chemical group at the 5′-end of the sense strand of thesiNA, or any other group that serves to render the sense strand inactiveas a guide sequence for mediating RNA interference. These modifications,for example, can result in a molecule where the 5′-end of the sensestrand no longer has a free 5′-hydroxyl (5′-OH) or a free 5′-phosphategroup (e.g., phosphate, diphosphate, triphosphate, cyclic phosphateetc.). Non-limiting examples of such siNA constructs are describedherein, such as “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”,“Stab 23/24”, “Stab 24/25”, and “Stab 24/26” (e.g., any siNA having Stab7, 9, 17, 23, or 24 sense strands) chemistries and variants thereof (seeTable IV) wherein the 5′-end and 3′-end of the sense strand of the siNAdo not comprise a hydroxyl group or phosphate group.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved specificity for down regulatingor inhibiting the expression of a target nucleic acid (e.g., a DNA orRNA such as a gene or its corresponding RNA), comprising introducing oneor more chemical modifications into the structure of a siNA moleculethat prevent a strand or portion of the siNA molecule from acting as atemplate or guide sequence for RNAi activity. In one embodiment, theinactive strand or sense region of the siNA molecule is the sense strandor sense region of the siNA molecule, i.e. the strand or region of thesiNA that does not have complementarity to the target nucleic acidsequence. In one embodiment, such chemical modifications comprise anychemical group at the 5′-end of the sense strand or region of the siNAthat does not comprise a 5′-hydroxyl (5′-OH) or 5′-phosphate group, orany other group that serves to render the sense strand or sense regioninactive as a guide sequence for mediating RNA interference.Non-limiting examples of such siNA constructs are described herein, suchas “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”, “Stab 23/24”,“Stab 24/25”, and “Stab 24/26” (e.g., any siNA having Stab 7, 9, 17, 23,or 24 sense strands) chemistries and variants thereof (see Table IV)wherein the 5′-end and 3′-end of the sense strand of the siNA do notcomprise a hydroxyl group or phosphate group.

In one embodiment, the invention features a method for screening siNAmolecules that are active in mediating RNA interference against a targetnucleic acid sequence comprising (a) generating a plurality ofunmodified siNA molecules, (b) screening the siNA molecules of step (a)under conditions suitable for isolating siNA molecules that are activein mediating RNA interference against the target nucleic acid sequence,and (c) introducing chemical modifications (e.g. chemical modificationsas described herein or as otherwise known in the art) into the activesiNA molecules of (b). In one embodiment, the method further comprisesre-screening the chemically modified siNA molecules of step (c) underconditions suitable for isolating chemically modified siNA moleculesthat are active in mediating RNA interference against the target nucleicacid sequence.

In one embodiment, the invention features a method for screeningchemically modified siNA molecules that are active in mediating RNAinterference against a target nucleic acid sequence comprising (a)generating a plurality of chemically modified siNA molecules (e.g. siNAmolecules as described herein or as otherwise known in the art), and (b)screening the siNA molecules of step (a) under conditions suitable forisolating chemically modified siNA molecules that are active inmediating RNA interference against the target nucleic acid sequence.

The term “ligand” refers to any compound or molecule, such as a drug,peptide, hormone, or neurotransmitter, that is capable of interactingwith another compound, such as a receptor, either directly orindirectly. The receptor that interacts with a ligand can be present onthe surface of a cell or can alternately be an intercellular receptor.Interaction of the ligand with the receptor can result in a biochemicalreaction, or can simply be a physical interaction or association.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing an excipient formulation to a siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchexcipients include polymers such as cyclodextrins, lipids, cationiclipids, polyamines, phospholipids, nanoparticles, receptors, ligands,and others.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing nucleotides having any of Formulae I-VII or anycombination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having improved bioavailability.

In another embodiment, polyethylene glycol (PEG) can be covalentlyattached to siNA compounds of the present invention. The attached PEGcan be any molecular weight, preferably from about 2,000 to about 50,000daltons (Da).

The present invention can be used alone or as a component of a kithaving at least one of the reagents necessary to carry out the in vitroor in vivo introduction of RNA to test samples and/or subjects. Forexample, preferred components of the kit include a siNA molecule of theinvention and a vehicle that promotes introduction of the siNA intocells of interest as described herein (e.g., using lipids and othermethods of transfection known in the art, see for example Beigelman etal, U.S. Pat. No. 6,395,713). The kit can be used for target validation,such as in determining gene function and/or activity, or in drugoptimization, and in drug discovery (see for example Usman et al., U.S.Ser. No. 60/402,996). Such a kit can also include instructions to allowa user of the kit to practice the invention.

The term “short interfering nucleic acid”, “siNA”, “short interferingRNA”, “siRNA”, “short interfering nucleic acid molecule”, “shortinterfering oligonucleotide molecule”, or “chemically-modified shortinterfering nucleic acid molecule” as used herein refers to any nucleicacid molecule capable of inhibiting or down regulating gene expressionor viral replication, for example by mediating RNA interference “RNAi”or gene silencing in a sequence-specific manner; see for example Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429;Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al.,International PCT Publication No. WO 00/44895; Zernicka-Goetz et al.,International PCT Publication No. WO 01/36646; Fire, International PCTPublication No. WO 99/32619; Plaetinck et al., International PCTPublication No. WO 00/01846; Mello and Fire, International PCTPublication No. WO 01/29058; Deschamps-Depaillette, International PCTPublication No. WO 99/07409; and Li et al., International PCTPublication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819;Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science,297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237;Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002,RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; andReinhart & Bartel, 2002, Science, 297, 1831). Non limiting examples ofsiNA molecules of the invention are shown in FIGS. 4-6, and Tables IIand III herein. For example the siNA can be a double-strandedpolynucleotide molecule comprising self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a targetnucleic acid molecule or a portion thereof and the sense region havingnucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof. The siNA can be assembled from two separateoligonucleotides, where one strand is the sense strand and the other isthe antisense strand, wherein the antisense and sense strands areself-complementary (i.e. each strand comprises nucleotide sequence thatis complementary to nucleotide sequence in the other strand; such aswhere the antisense strand and sense strand form a duplex or doublestranded structure, for example wherein the double stranded region isabout 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprisesnucleotide sequence that is complementary to nucleotide sequence in atarget nucleic acid molecule or a portion thereof and the sense strandcomprises nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof (e.g., about 15 to about 25 or morenucleotides of the siNA molecule are complementary to the target nucleicacid or a portion thereof). Alternatively, the siNA is assembled from asingle oligonucleotide, where the self-complementary sense and antisenseregions of the siNA are linked by means of a nucleic acid based ornon-nucleic acid-based linker(s). The siNA can be a polynucleotide witha duplex, asymmetric duplex, hairpin or asymmetric hairpin secondarystructure, having self-complementary sense and antisense regions,wherein the antisense region comprises nucleotide sequence that iscomplementary to nucleotide sequence in a separate target nucleic acidmolecule or a portion thereof and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof. The siNA can be a circular single-stranded polynucleotidehaving two or more loop structures and a stem comprisingself-complementary sense and antisense regions, wherein the antisenseregion comprises nucleotide sequence that is complementary to nucleotidesequence in a target nucleic acid molecule or a portion thereof and thesense region having nucleotide sequence corresponding to the targetnucleic acid sequence or a portion thereof, and wherein the circularpolynucleotide can be processed either in vivo or in vitro to generatean active siNA molecule capable of mediating RNAi. The siNA can alsocomprise a single stranded polynucleotide having nucleotide sequencecomplementary to nucleotide sequence in a target nucleic acid moleculeor a portion thereof (for example, where such siNA molecule does notrequire the presence within the siNA molecule of nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof),wherein the single stranded polynucleotide can further comprise aterminal phosphate group, such as a 5′-phosphate (see for exampleMartinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002,Molecular Cell, 10, 537-568), or 5′,3′-diphosphate. In certainembodiments, the siNA molecule of the invention comprises separate senseand antisense sequences or regions, wherein the sense and antisenseregions are covalently linked by nucleotide or non-nucleotide linkersmolecules as is known in the art, or are alternately non-covalentlylinked by ionic interactions, hydrogen bonding, van der waalsinteractions, hydrophobic interactions, and/or stacking interactions. Incertain embodiments, the siNA molecules of the invention comprisenucleotide sequence that is complementary to nucleotide sequence of atarget gene. In another embodiment, the siNA molecule of the inventioninteracts with nucleotide sequence of a target gene in a manner thatcauses inhibition of expression of the target gene. As used herein, siNAmolecules need not be limited to those molecules containing only RNA,but further encompasses chemically-modified nucleotides andnon-nucleotides. In certain embodiments, the short interfering nucleicacid molecules of the invention lack 2′-hydroxy (2′-OH) containingnucleotides. Applicant describes in certain embodiments shortinterfering nucleic acids that do not require the presence ofnucleotides having a 2′-hydroxy group for mediating RNAi and as such,short interfering nucleic acid molecules of the invention optionally donot include any ribonucleotides (e.g., nucleotides having a 2′-OHgroup). Such siNA molecules that do not require the presence ofribonucleotides within the siNA molecule to support RNAi can howeverhave an attached linker or linkers or other attached or associatedgroups, moieties, or chains containing one or more nucleotides with2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides atabout 5, 10, 20, 30, 40, or 50% of the nucleotide positions. Themodified short interfering nucleic acid molecules of the invention canalso be referred to as short interfering modified oligonucleotides“siMON.” As used herein, the term siNA is meant to be equivalent toother terms used to describe nucleic acid molecules that are capable ofmediating sequence specific RNAi, for example short interfering RNA(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpinRNA (shRNA), short interfering oligonucleotide, short interferingnucleic acid, short interfering modified oligonucleotide,chemically-modified siRNA, post-transcriptional gene silencing RNA(ptgsRNA), and others. In addition, as used herein, the term RNAi ismeant to be equivalent to other terms used to describe sequence specificRNA interference, such as post transcriptional gene silencing,translational inhibition, or epigenetics. For example, siNA molecules ofthe invention can be used to epigenetically silence genes at both thepost-transcriptional level or the pre-transcriptional level. In anon-limiting example, epigenetic regulation of gene expression by siNAmolecules of the invention can result from siNA mediated modification ofchromatin structure or methylation pattern to alter gene expression(see, for example, Verdel et al., 2004, Science, 303, 672-676;Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science,297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,2232-2237).

In one embodiment, a siNA molecule of the invention is a duplex formingoligonucleotide “DFO”, (see for example FIGS. 14-15 and Vaish et al.,U.S. Ser. No. 10/727,780 filed Dec. 3, 2003 and International PCTApplication No. US04/16390, filed May 24, 2004).

In one embodiment, a siNA molecule of the invention is a multifunctionalsiNA, (see for example FIGS. 16-21 and Jadhav et al., U.S. Ser. No.60/543,480 filed Feb. 10, 2004 and International PCT Application No.US04/16390, filed May 24, 2004). The multifunctional siNA of theinvention can comprise sequence targeting, for example, two regions ofSCD RNA (see for example target sequences in Tables II and III).

By “asymmetric hairpin” as used herein is meant a linear siNA moleculecomprising an antisense region, a loop portion that can comprisenucleotides or non-nucleotides, and a sense region that comprises fewernucleotides than the antisense region to the extent that the senseregion has enough complementary nucleotides to base pair with theantisense region and form a duplex with loop. For example, an asymmetrichairpin siNA molecule of the invention can comprise an antisense regionhaving length sufficient to mediate RNAi in a cell or in vitro system(e.g. about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 nucleotides) and a loop region comprisingabout 4 to about 12 (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, or 12)nucleotides, and a sense region having about 3 to about 25 (e.g., about3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25) nucleotides that are complementary to the antisenseregion. The asymmetric hairpin siNA molecule can also comprise a5′-terminal phosphate group that can be chemically modified. The loopportion of the asymmetric hairpin siNA molecule can comprisenucleotides, non-nucleotides, linker molecules, or conjugate moleculesas described herein.

By “asymmetric duplex” as used herein is meant a siNA molecule havingtwo separate strands comprising a sense region and an antisense region,wherein the sense region comprises fewer nucleotides than the antisenseregion to the extent that the sense region has enough complementarynucleotides to base pair with the antisense region and form a duplex.For example, an asymmetric duplex siNA molecule of the invention cancomprise an antisense region having length sufficient to mediate RNAi ina cell or in vitro system (e.g. about 15 to about 30, or about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides)and a sense region having about 3 to about 25 (e.g., about 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or25) nucleotides that are complementary to the antisense region.

By “modulate” is meant that the expression of the gene, or level of RNAmolecule or equivalent RNA molecules encoding one or more proteins orprotein subunits, or activity of one or more proteins or proteinsubunits is up regulated or down regulated, such that expression, level,or activity is greater than or less than that observed in the absence ofthe modulator. For example, the term “modulate” can mean “inhibit,” butthe use of the word “modulate” is not limited to this definition.

By “inhibit”, “down-regulate”, or “reduce”, it is meant that theexpression of the gene, or level of RNA molecules or equivalent RNAmolecules encoding one or more proteins or protein subunits, or activityof one or more proteins or protein subunits, is reduced below thatobserved in the absence of the nucleic acid molecules (e.g., siNA) ofthe invention. In one embodiment, inhibition, down-regulation orreduction with an siNA molecule is below that level observed in thepresence of an inactive or attenuated molecule. In another embodiment,inhibition, down-regulation, or reduction with siNA molecules is belowthat level observed in the presence of, for example, an siNA moleculewith scrambled sequence or with mismatches. In another embodiment,inhibition, down-regulation, or reduction of gene expression with anucleic acid molecule of the instant invention is greater in thepresence of the nucleic acid molecule than in its absence. In oneembodiment, inhibition, down regulation, or reduction of gene expressionis associated with post transcriptional silencing, such as RNAi mediatedcleavage of a target nucleic acid molecule (e.g. RNA) or inhibition oftranslation. In one embodiment, inhibition, down regulation, orreduction of gene expression is associated with pretranscriptionalsilencing.

By “gene”, or “target gene”, is meant a nucleic acid that encodes anRNA, for example, nucleic acid sequences including, but not limited to,structural genes encoding a polypeptide. A gene or target gene can alsoencode a functional RNA (fRNA) or non-coding RNA (ncRNA), such as smalltemporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA),short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomalRNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Suchnon-coding RNAs can serve as target nucleic acid molecules for siNAmediated RNA interference in modulating the activity of fRNA or ncRNAinvolved in functional or regulatory cellular processes. Abberant fRNAor ncRNA activity leading to disease can therefore be modulated by siNAmolecules of the invention. siNA molecules targeting fRNA and ncRNA canalso be used to manipulate or alter the genotype or phenotype of asubject, organism or cell, by intervening in cellular processes such asgenetic imprinting, transcription, translation, or nucleic acidprocessing (e.g., transamination, methylation etc.). The target gene canbe a gene derived from a cell, an endogenous gene, a transgene, orexogenous genes such as genes of a pathogen, for example a virus, whichis present in the cell after infection thereof. The cell containing thetarget gene can be derived from or contained in any organism, forexample a plant, animal, protozoan, virus, bacterium, or fungus.Non-limiting examples of plants include monocots, dicots, orgymnosperms. Non-limiting examples of animals include vertebrates orinvertebrates. Non-limiting examples of fungi include molds or yeasts.For a review, see for example Snyder and Gerstein, 2003, Science, 300,258-260.

By “non-canonical base pair” is meant any non-Watson Crick base pair,such as mismatches and/or wobble base pairs, including flippedmismatches, single hydrogen bond mismatches, trans-type mismatches,triple base interactions, and quadruple base interactions. Non-limitingexamples of such non-canonical base pairs include, but are not limitedto, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AAN7 amino, CC 2-carbonyl-amino(H1)-N3-amino(H2), GA sheared, UC4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AUreverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AAN1-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl,GA+carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-aminosymmetric, CC carbonyl-amino symmetric, CC N3-amino symmetric, UU2-carbonyl-imino symmetric, UU 4-carbonyl-imino symmetric, AA amino-N3,AA N1-amino, AC amino 2-carbonyl, AC N3-amino, AC N7-amino, AUamino-4-carbonyl, AU N1-imino, AU N3-imino, AU N7-imino, CCcarbonyl-amino, GA amino-N1, GA amino-N7, GA carbonyl-amino, GAN3-amino, GC amino-N3, GC carbonyl-amino, GC N3-amino, GC N7-amino, GGamino-N7, GG carbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GUcarbonyl-imino, GU imino-2-carbonyl, GU N7-imino, psiU imino-2-carbonyl,UC 4-carbonyl-amino, UC imino-carbonyl, UU imino-4-carbonyl, AC C2-H-N3,GA carbonyl-C2-H, UU imino-4-carbonyl 2 carbonyl-C5-H, AC amino(A)N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonyl amino-2-carbonyl, and GU imino amino-2-carbonyl base pairs.

By “SCD” as used herein is meant, any stearoyl-CoA desaturase protein,peptide, or polypeptide having any stearoyl-CoA desaturase activity,such as encoded by SCD Genbank Accession Nos. shown in Table I. The termSCD also refers to nucleic acid sequences encoding any stearoyl-CoAdesaturase protein, peptide, or polypeptide having SCD activity. Theterm “SCD” is also meant to include other SCD encoding sequence, such asother SCD isoforms, mutant SCD genes, splice variants of SCD genes, andSCD gene polymorphisms.

By “homologous sequence” is meant, a nucleotide sequence that is sharedby one or more polynucleotide sequences, such as genes, gene transcriptsand/or non-coding polynucleotides. For example, a homologous sequencecan be a nucleotide sequence that is shared by two or more genesencoding related but different proteins, such as different members of agene family, different protein epitopes, different protein isoforms orcompletely divergent genes, such as a cytokine and its correspondingreceptors. A homologous sequence can be a nucleotide sequence that isshared by two or more non-coding polynucleotides, such as noncoding DNAor RNA, regulatory sequences, introns, and sites of transcriptionalcontrol or regulation. Homologous sequences can also include conservedsequence regions shared by more than one polynucleotide sequence.Homology does not need to be perfect homology (e.g., 100%), as partiallyhomologous sequences are also contemplated by the instant invention(e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%,86%, 85%, 84%, 83%, 82%, 81%, 80% etc.).

By “conserved sequence region” is meant, a nucleotide sequence of one ormore regions in a polynucleotide does not vary significantly betweengenerations or from one biological system, subject, or organism toanother biological system, subject, or organism. The polynucleotide caninclude both coding and non-coding DNA and RNA.

By “sense region” is meant a nucleotide sequence of a siNA moleculehaving complementarity to an antisense region of the siNA molecule. Inaddition, the sense region of a siNA molecule can comprise a nucleicacid sequence having homology with a target nucleic acid sequence.

By “antisense region” is meant a nucleotide sequence of a siNA moleculehaving complementarity to a target nucleic acid sequence. In addition,the antisense region of a siNA molecule can optionally comprise anucleic acid sequence having complementarity to a sense region of thesiNA molecule.

By “target nucleic acid” is meant any nucleic acid sequence whoseexpression or activity is to be modulated. The target nucleic acid canbe DNA or RNA.

By “complementarity” is meant that a nucleic acid can form hydrogenbond(s) with another nucleic acid sequence by either traditionalWatson-Crick or other non-traditional types. In reference to the nucleicmolecules of the present invention, the binding free energy for anucleic acid molecule with its complementary sequence is sufficient toallow the relevant function of the nucleic acid to proceed, e.g., RNAiactivity. Determination of binding free energies for nucleic acidmolecules is well known in the art (see, e.g., Turner et al., 1987, CSHSymp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad.Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.109:3783-3785). A percent complementarity indicates the percentage ofcontiguous residues in a nucleic acid molecule that can form hydrogenbonds (e.g., Watson-Crick base pairing) with a second nucleic acidsequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10nucleotides in the first oligonucleotide being based paired to a secondnucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%,80%, 90%, and 100% complementary respectively). “Perfectlycomplementary” means that all the contiguous residues of a nucleic acidsequence will hydrogen bond with the same number of contiguous residuesin a second nucleic acid sequence. In one embodiment, a siNA molecule ofthe invention comprises about 15 to about 30 or more (e.g., about 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more)nucleotides that are complementary to one or more target nucleic acidmolecules or a portion thereof.

In one embodiment, siNA molecules of the invention that down regulate orreduce SCD gene expression are used for preventing or treating diabetes(type I and/or type II), atherosclerosis, cancer, obesity, and viralinfection in a subject or organism.

In one embodiment, the siNA molecules of the invention are used to treatdiabetes (type I and/or type II), atherosclerosis, cancer, obesity, andviral infection in a subject or organism.

In one embodiment, the siNA molecules of the invention are used toinduce or promote weight loss in a subject or organism. In oneembodiment, the siNA molecules of the invention are used to lower theamount of body fat in a subject or organism.

By “cancer” as used herein is meant, any disease, condition, trait,genotype or phenotype characterized by unregulated cell growth orreplication as is known in the art; including AIDS related cancers suchas Kaposi's sarcoma; breast cancers; bone cancers such as Osteosarcoma,Chondrosarcomas, Ewing's sarcoma, Fibrosarcomas, Giant cell tumors,Adamantinomas, and Chordomas; Brain cancers such as Meningiomas,Glioblastomas, Lower-Grade Astrocytomas, Oligodendrocytomas, PituitaryTumors, Schwannomas, and Metastatic brain cancers; cancers of the headand neck including various lymphomas such as mantle cell lymphoma,non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngealcarcinoma, gallbladder and bile duct cancers, cancers of the retina suchas retinoblastoma, cancers of the esophagus, gastric cancers, multiplemyeloma, ovarian cancer, uterine cancer, thyroid cancer, testicularcancer, endometrial cancer, melanoma, colorectal cancer, lung cancer,bladder cancer, prostate cancer, lung cancer (including non-small celllung carcinoma), pancreatic cancer, sarcomas, Wilms' tumor, cervicalcancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma,liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladderadeno carcinoma, parotid adenocarcinoma, endometrial sarcoma, multidrugresistant cancers; and any other cancer that can respond to themodulation of disease related gene expression in a cell or tissue, aloneor in combination with other therapies.

In one embodiment of the present invention, each sequence of a siNAmolecule of the invention is independently about 15 to about 30nucleotides in length, in specific embodiments about 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. Inanother embodiment, the siNA duplexes of the invention independentlycomprise about 15 to about 30 base pairs (e.g., about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In anotherembodiment, one or more strands of the siNA molecule of the inventionindependently comprises about 15 to about 30 nucleotides (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) thatare complementary to a target nucleic acid molecule. In yet anotherembodiment, siNA molecules of the invention comprising hairpin orcircular structures are about 35 to about 55 (e.g., about 35, 40, 45, 50or 55) nucleotides in length, or about 38 to about 44 (e.g., about 38,39, 40, 41, 42, 43, or 44) nucleotides in length and comprising about 15to about 25 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25)base pairs. Exemplary siNA molecules of the invention are shown in TableII. Exemplary synthetic siNA molecules of the invention are shown inTable III and/or FIGS. 4-5.

As used herein “cell” is used in its usual biological sense, and doesnot refer to an entire multicellular organism, e.g., specifically doesnot refer to a human. The cell can be present in an organism, e.g.,birds, plants and mammals such as humans, cows, sheep, apes, monkeys,swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterialcell) or eukaryotic (e.g., mammalian or plant cell). The cell can be ofsomatic or germ line origin, totipotent or pluripotent, dividing ornon-dividing. The cell can also be derived from or can comprise a gameteor embryo, a stem cell, or a fully differentiated cell.

The siNA molecules of the invention are added directly, or can becomplexed with cationic lipids, packaged within liposomes, or otherwisedelivered to target cells or tissues. The nucleic acid or nucleic acidcomplexes can be locally administered to relevant tissues ex vivo, or invivo through direct dermal application, transdermal application, orinjection, with or without their incorporation in biopolymers. Inparticular embodiments, the nucleic acid molecules of the inventioncomprise sequences shown in Tables II-III and/or FIGS. 4-5. Examples ofsuch nucleic acid molecules consist essentially of sequences defined inthese tables and figures. Furthermore, the chemically modifiedconstructs described in Table IV can be applied to any siNA sequence ofthe invention.

In another aspect, the invention provides mammalian cells containing oneor more siNA molecules of this invention. The one or more siNA moleculescan independently be targeted to the same or different sites.

By “RNA” is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of a β-D-ribofuranose moiety. The terms includedouble-stranded RNA, single-stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient ofexplanted cells or the cells themselves. “Subject” also refers to anorganism to which the nucleic acid molecules of the invention can beadministered. A subject can be a mammal or mammalian cells, including ahuman or human cells.

The term “phosphorothioate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise a sulfur atom.Hence, the term phosphorothioate refers to both phosphorothioate andphosphorodithioate internucleotide linkages.

The term “phosphonoacetate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise an acetyl orprotected acetyl group.

The term “thiophosphonoacetate” as used herein refers to aninternucleotide linkage having Formula I, wherein Z comprises an acetylor protected acetyl group and W comprises a sulfur atom or alternately Wcomprises an acetyl or protected acetyl group and Z comprises a sulfuratom.

The term “universal base” as used herein refers to nucleotide baseanalogs that form base pairs with each of the natural DNA/RNA bases withlittle discrimination between them. Non-limiting examples of universalbases include C-phenyl, C-naphthyl and other aromatic derivatives,inosine, azole carboxamides, and nitroazole derivatives such as3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as knownin the art (see for example Loakes, 2001, Nucleic Acids Research, 29,2437-2447).

The term “acyclic nucleotide” as used herein refers to any nucleotidehaving an acyclic ribose sugar, for example where any of the ribosecarbons (C1, C2, C3, C4, or C5), are independently or in combinationabsent from the nucleotide.

The nucleic acid molecules of the instant invention, individually, or incombination or in conjunction with other drugs, can be used to forpreventing or treating diabetes (type I and/or type II),atherosclerosis, cancer, obesity, and viral infection in a subject ororganism.

For example, the siNA molecules can be administered to a subject or canbe administered to other appropriate cells evident to those skilled inthe art, individually or in combination with one or more drugs underconditions suitable for the treatment.

In a further embodiment, the siNA molecules can be used in combinationwith other known treatments to prevent or treat diabetes (type I and/ortype II), atherosclerosis, cancer, obesity, and viral infection in asubject or organism. For example, the described molecules could be usedin combination with one or more known compounds, treatments, orprocedures to prevent or treat diabetes (type I and/or type II),atherosclerosis, cancer, obesity, and viral infection in a subject ororganism as are known in the art.

In one embodiment, the invention features an expression vectorcomprising a nucleic acid sequence encoding at least one siNA moleculeof the invention, in a manner which allows expression of the siNAmolecule. For example, the vector can contain sequence(s) encoding bothstrands of a siNA molecule comprising a duplex. The vector can alsocontain sequence(s) encoding a single nucleic acid molecule that isself-complementary and thus forms a siNA molecule. Non-limiting examplesof such expression vectors are described in Paul et al., 2002, NatureBiotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology,19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina etal., 2002, Nature Medicine, advance online publicationdoi:10.1038/nm725.

In another embodiment, the invention features a mammalian cell, forexample, a human cell, including an expression vector of the invention.

In yet another embodiment, the expression vector of the inventioncomprises a sequence for a siNA molecule having complementarity to a RNAmolecule referred to by a Genbank Accession numbers, for example GenbankAccession Nos. shown in Table I.

In one embodiment, an expression vector of the invention comprises anucleic acid sequence encoding two or more siNA molecules, which can bethe same or different.

In another aspect of the invention, siNA molecules that interact withtarget RNA molecules and down-regulate gene encoding target RNAmolecules (for example target RNA molecules referred to by GenbankAccession numbers herein) are expressed from transcription unitsinserted into DNA or RNA vectors. The recombinant vectors can be DNAplasmids or viral vectors. siNA expressing viral vectors can beconstructed based on, but not limited to, adeno-associated virus,retrovirus, adenovirus, or alphavirus. The recombinant vectors capableof expressing the siNA molecules can be delivered as described herein,and persist in target cells. Alternatively, viral vectors can be usedthat provide for transient expression of siNA molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siNAmolecules bind and down-regulate gene function or expression via RNAinterference (RNAi). Delivery of siNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from a subject followed byreintroduction into the subject, or by any other means that would allowfor introduction into the desired target cell.

By “vectors” is meant any nucleic acid- and/or viral-based techniqueused to deliver a desired nucleic acid.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a non-limiting example of a scheme for the synthesis ofsiNA molecules. The complementary siNA sequence strands, strand 1 andstrand 2, are synthesized in tandem and are connected by a cleavablelinkage, such as a nucleotide succinate or abasic succinate, which canbe the same or different from the cleavable linker used for solid phasesynthesis on a solid support. The synthesis can be either solid phase orsolution phase, in the example shown, the synthesis is a solid phasesynthesis.

The synthesis is performed such that a protecting group, such as adimethoxytrityl group, remains intact on the terminal nucleotide of thetandem oligonucleotide. Upon cleavage and deprotection of theoligonucleotide, the two siNA strands spontaneously hybridize to form asiNA duplex, which allows the purification of the duplex by utilizingthe properties of the terminal protecting group, for example by applyinga trityl on purification method wherein only duplexes/oligonucleotideswith the terminal protecting group are isolated.

FIG. 2 shows a MALDI-TOF mass spectrum of a purified siNA duplexsynthesized by a method of the invention. The two peaks shown correspondto the predicted mass of the separate siNA sequence strands. This resultdemonstrates that the siNA duplex generated from tandem synthesis can bepurified as a single entity using a simple trityl-on purificationmethodology.

FIG. 3 shows a non-limiting proposed mechanistic representation oftarget RNA degradation involved in RNAi. Double-stranded RNA (dsRNA),which is generated by RNA-dependent RNA polymerase (RdRP) from foreignsingle-stranded RNA, for example viral, transposon, or other exogenousRNA, activates the DICER enzyme that in turn generates siNA duplexes.Alternately, synthetic or expressed siNA can be introduced directly intoa cell by appropriate means. An active siNA complex forms whichrecognizes a target RNA, resulting in degradation of the target RNA bythe RISC endonuclease complex or in the synthesis of additional RNA byRNA-dependent RNA polymerase (RdRP), which can activate DICER and resultin additional siNA molecules, thereby amplifying the RNAi response.

FIG. 4A-F shows non-limiting examples of chemically-modified siNAconstructs of the present invention. In the figure, N stands for anynucleotide (adenosine, guanosine, cytosine, uridine, or optionallythymidine, for example thymidine can be substituted in the overhangingregions designated by parenthesis (N N). Various modifications are shownfor the sense and antisense strands of the siNA constructs.

FIG. 4A: The sense strand comprises 21 nucleotides wherein the twoterminal 3′-nucleotides are optionally base paired and wherein allnucleotides present are ribonucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. The antisense strandcomprises 21 nucleotides, optionally having a 3′-terminal glycerylmoiety wherein the two terminal 3′-nucleotides are optionallycomplementary to the target RNA sequence, and wherein all nucleotidespresent are ribonucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”,optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4B: The sense strand comprises 21 nucleotides wherein the twoterminal 3′-nucleotides are optionally base paired and wherein allpyrimidine nucleotides that may be present are 2′deoxy-2′-fluoromodified nucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. The antisense strand comprises21 nucleotides, optionally having a 3′-terminal glyceryl moiety andwherein the two terminal 3′-nucleotides are optionally complementary tothe target RNA sequence, and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides and all purinenucleotides that may be present are 2′-O-methyl modified nucleotidesexcept for (N N) nucleotides, which can comprise ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. A modified internucleotide linkage, such as aphosphorothioate, phosphorodithioate or other modified internucleotidelinkage as described herein, shown as “s”, optionally connects the (N N)nucleotides in the sense and antisense strand.

FIG. 4C: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotidesexcept for (N N) nucleotides, which can comprise ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. The antisense strand comprises 21 nucleotides,optionally having a 3′-terminal glyceryl moiety and wherein the twoterminal 3′-nucleotides are optionally complementary to the target RNAsequence, and wherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. A modifiedinternucleotide linkage, such as a phosphorothioate, phosphorodithioateor other modified internucleotide linkage as described herein, shown as“s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4D: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein andwherein and all purine nucleotides that may be present are 2′-deoxynucleotides. The antisense strand comprises 21 nucleotides, optionallyhaving a 3′-terminal glyceryl moiety and wherein the two terminal3′-nucleotides are optionally complementary to the target RNA sequence,wherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides thatmay be present are 2′-O-methyl modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Amodified internucleotide linkage, such as a phosphorothioate,phosphorodithioate or other modified internucleotide linkage asdescribed herein, shown as “s”, optionally connects the (N N)nucleotides in the antisense strand.

FIG. 4E: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Theantisense strand comprises 21 nucleotides, optionally having a3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotidesare optionally complementary to the target RNA sequence, and wherein allpyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoromodified nucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”,optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4F: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein andwherein and all purine nucleotides that may be present are 2′-deoxynucleotides. The antisense strand comprises 21 nucleotides, optionallyhaving a 3′-terminal glyceryl moiety and wherein the two terminal3′-nucleotides are optionally complementary to the target RNA sequence,and having one 3′-terminal phosphorothioate internucleotide linkage andwherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides thatmay be present are 2′-deoxy nucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. A modifiedinternucleotide linkage, such as a phosphorothioate, phosphorodithioateor other modified internucleotide linkage as described herein, shown as“s”, optionally connects the (N N) nucleotides in the antisense strand.The antisense strand of constructs A-F comprise sequence complementaryto any target nucleic acid sequence of the invention. Furthermore, whena glyceryl moiety (L) is present at the 3′-end of the antisense strandfor any construct shown in FIG. 4A-F, the modified internucleotidelinkage is optional.

FIG. 5A-F shows non-limiting examples of specific chemically-modifiedsiNA sequences of the invention. A-F applies the chemical modificationsdescribed in FIG. 4A-F to a SCD siNA sequence. Such chemicalmodifications can be applied to any SCD sequence and/or SCD polymorphismsequence.

FIG. 6 shows non-limiting examples of different siNA constructs of theinvention. The examples shown (constructs 1, 2, and 3) have 19representative base pairs; however, different embodiments of theinvention include any number of base pairs described herein. Bracketedregions represent nucleotide overhangs, for example, comprising about 1,2, 3, or 4 nucleotides in length, preferably about 2 nucleotides.Constructs 1 and 2 can be used independently for RNAi activity.Construct 2 can comprise a polynucleotide or non-nucleotide linker,which can optionally be designed as a biodegradable linker. In oneembodiment, the loop structure shown in construct 2 can comprise abiodegradable linker that results in the formation of construct 1 invivo and/or in vitro. In another example, construct 3 can be used togenerate construct 2 under the same principle wherein a linker is usedto generate the active siNA construct 2 in vivo and/or in vitro, whichcan optionally utilize another biodegradable linker to generate theactive siNA construct 1 in vivo and/or in vitro. As such, the stabilityand/or activity of the siNA constructs can be modulated based on thedesign of the siNA construct for use in vivo or in vitro and/or invitro.

FIG. 7A-C is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate siNA hairpin constructs.

FIG. 7A: A DNA oligomer is synthesized with a 5′-restriction site (R1)sequence followed by a region having sequence identical (sense region ofsiNA) to a predetermined SCD target sequence, wherein the sense regioncomprises, for example, about 19, 20, 21, or 22 nucleotides (N) inlength, which is followed by a loop sequence of defined sequence (X),comprising, for example, about 3 to about 10 nucleotides.

FIG. 7B: The synthetic construct is then extended by DNA polymerase togenerate a hairpin structure having self-complementary sequence thatwill result in a siNA transcript having specificity for a SCD targetsequence and having self-complementary sense and antisense regions.

FIG. 7C: The construct is heated (for example to about 95° C) tolinearize the sequence, thus allowing extension of a complementarysecond DNA strand using a primer to the 3′-restriction sequence of thefirst strand. The double-stranded DNA is then inserted into anappropriate vector for expression in cells. The construct can bedesigned such that a 3′-terminal nucleotide overhang results from thetranscription, for example, by engineering restriction sites and/orutilizing a poly-U termination region as described in Paul et al., 2002,Nature Biotechnology, 29, 505-508.

FIG. 8A-C is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate double-stranded siNAconstructs.

FIG. 8A: A DNA oligomer is synthesized with a 5′-restriction (R1) sitesequence followed by a region having sequence identical (sense region ofsiNA) to a predetermined SCD target sequence, wherein the sense regioncomprises, for example, about 19, 20, 21, or 22 nucleotides (N) inlength, and which is followed by a 3′-restriction site (R2) which isadjacent to a loop sequence of defined sequence (X).

FIG. 8B: The synthetic construct is then extended by DNA polymerase togenerate a hairpin structure having self-complementary sequence.

FIG. 8C: The construct is processed by restriction enzymes specific toR1 and R2 to generate a double-stranded DNA which is then inserted intoan appropriate vector for expression in cells. The transcriptioncassette is designed such that a U6 promoter region flanks each side ofthe dsDNA which generates the separate sense and antisense strands ofthe siNA. Poly T termination sequences can be added to the constructs togenerate U overhangs in the resulting transcript.

FIG. 9A-E is a diagrammatic representation of a method used to determinetarget sites for siNA mediated RNAi within a particular target nucleicacid sequence, such as messenger RNA.

FIG. 9A: A pool of siNA oligonucleotides are synthesized wherein theantisense region of the siNA constructs has complementarity to targetsites across the target nucleic acid sequence, and wherein the senseregion comprises sequence complementary to the antisense region of thesiNA.

FIGS. 9B&C: (FIG. 9B) The sequences are pooled and are inserted intovectors such that (FIG. 9C) transfection of a vector into cells resultsin the expression of the siNA.

FIG. 9D: Cells are sorted based on phenotypic change that is associatedwith modulation of the target nucleic acid sequence.

FIG. 9E: The siNA is isolated from the sorted cells and is sequenced toidentify efficacious target sites within the target nucleic acidsequence.

FIG. 10 shows non-limiting examples of different stabilizationchemistries (1-10) that can be used, for example, to stabilize the3′-end of siNA sequences of the invention, including (1) [3-3′]-inverteddeoxyribose; (2) deoxyribonucleotide; (3)[5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5)[5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7)[3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9)[5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide. Inaddition to modified and unmodified backbone chemistries indicated inthe figure, these chemistries can be combined with different backbonemodifications as described herein, for example, backbone modificationshaving Formula I. In addition, the 2′-deoxy nucleotide shown 5′ to theterminal modifications shown can be another modified or unmodifiednucleotide or non-nucleotide described herein, for example modificationshaving any of Formulae I-VII or any combination thereof.

FIG. 11 shows a non-limiting example of a strategy used to identifychemically modified siNA constructs of the invention that are nucleaseresistance while preserving the ability to mediate RNAi activity.Chemical modifications are introduced into the siNA construct based oneducated design parameters (e.g. introducing 2′-mofications, basemodifications, backbone modifications, terminal cap modifications etc).The modified construct in tested in an appropriate system (e.g. humanserum for nuclease resistance, shown, or an animal model for PK/deliveryparameters). In parallel, the siNA construct is tested for RNAiactivity, for example in a cell culture system such as a luciferasereporter assay). Lead siNA constructs are then identified which possessa particular characteristic while maintaining RNAi activity, and can befurther modified and assayed once again. This same approach can be usedto identify siNA-conjugate molecules with improved pharmacokineticprofiles, delivery, and RNAi activity.

FIG. 12 shows non-limiting examples of phosphorylated siNA molecules ofthe invention, including linear and duplex constructs and asymmetricderivatives thereof.

FIG. 13 shows non-limiting examples of chemically modified terminalphosphate groups of the invention.

FIG. 14A shows a non-limiting example of methodology used to design selfcomplementary DFO constructs utilizing palindrome and/or repeat nucleicacid sequences that are identified in a target nucleic acid sequence.(i) A palindrome or repeat sequence is identified in a nucleic acidtarget sequence. (ii) A sequence is designed that is complementary tothe target nucleic acid sequence and the palindrome sequence. (iii) Aninverse repeat sequence of the non-palindrome/repeat portion of thecomplementary sequence is appended to the 3′-end of the complementarysequence to generate a self complementary DFO molecule comprisingsequence complementary to the nucleic acid target. (iv) The DFO moleculecan self-assemble to form a double stranded oligonucleotide. FIG. 14Bshows a non-limiting representative example of a duplex formingoligonucleotide sequence. FIG. 14C shows a non-limiting example of theself assembly schematic of a representative duplex formingoligonucleotide sequence. FIG. 14D shows a non-limiting example of theself assembly schematic of a representative duplex formingoligonucleotide sequence followed by interaction with a target nucleicacid sequence resulting in modulation of gene expression.

FIG. 15 shows a non-limiting example of the design of self complementaryDFO constructs utilizing palindrome and/or repeat nucleic acid sequencesthat are incorporated into the DFO constructs that have sequencecomplementary to any target nucleic acid sequence of interest.Incorporation of these palindrome/repeat sequences allow the design ofDFO constructs that form duplexes in which each strand is capable ofmediating modulation of target gene expression, for example by RNAi.First, the target sequence is identified. A complementary sequence isthen generated in which nucleotide or non-nucleotide modifications(shown as X or Y) are introduced into the complementary sequence thatgenerate an artificial palindrome (shown as XYXYXY in the Figure). Aninverse repeat of the non-palindrome/repeat complementary sequence isappended to the 3′-end of the complementary sequence to generate a selfcomplementary DFO comprising sequence complementary to the nucleic acidtarget. The DFO can self-assemble to form a double strandedoligonucleotide.

FIG. 16 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising two separate polynucleotide sequences that areeach capable of mediating RNAi directed cleavage of differing targetnucleic acid sequences. FIG. 16A shows a non-limiting example of amultifunctional siNA molecule having a first region that iscomplementary to a first target nucleic acid sequence (complementaryregion 1) and a second region that is complementary to a second targetnucleic acid sequence (complementary region 2), wherein the first andsecond complementary regions are situated at the 3′-ends of eachpolynucleotide sequence in the multifunctional siNA. The dashed portionsof each polynucleotide sequence of the multifunctional siNA constructhave complementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. FIG. 16B shows a non-limiting example of a multifunctionalsiNA molecule having a first region that is complementary to a firsttarget nucleic acid sequence (complementary region 1) and a secondregion that is complementary to a second target nucleic acid sequence(complementary region 2), wherein the first and second complementaryregions are situated at the 5′-ends of each polynucleotide sequence inthe multifunctional siNA. The dashed portions of each polynucleotidesequence of the multifunctional siNA construct have complementarity withregard to corresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences.

FIG. 17 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising a single polynucleotide sequence comprisingdistinct regions that are each capable of mediating RNAi directedcleavage of differing target nucleic acid sequences. FIG. 17A shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe second complementary region is situated at the 3′-end of thepolynucleotide sequence in the multifunctional siNA. The dashed portionsof each polynucleotide sequence of the multifunctional siNA constructhave complementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. FIG. 17B shows a non-limiting example of a multifunctionalsiNA molecule having a first region that is complementary to a firsttarget nucleic acid sequence (complementary region 1) and a secondregion that is complementary to a second target nucleic acid sequence(complementary region 2), wherein the first complementary region issituated at the 5′-end of the polynucleotide sequence in themultifunctional siNA. The dashed portions of each polynucleotidesequence of the multifunctional siNA construct have complementarity withregard to corresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. In one embodiment,these multifunctional siNA constructs are processed in vivo or in vitroto generate multifunctional siNA constructs as shown in FIG. 16.

FIG. 18 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising two separate polynucleotide sequences that areeach capable of mediating RNAi directed cleavage of differing targetnucleic acid sequences and wherein the multifunctional siNA constructfurther comprises a self complementary, palindrome, or repeat region,thus enabling shorter bifuctional siNA constructs that can mediate RNAinterference against differing target nucleic acid sequences. FIG. 18Ashows a non-limiting example of a multifunctional siNA molecule having afirst region that is complementary to a first target nucleic acidsequence (complementary region 1) and a second region that iscomplementary to a second target nucleic acid sequence (complementaryregion 2), wherein the first and second complementary regions aresituated at the 3′-ends of each polynucleotide sequence in themultifunctional siNA, and wherein the first and second complementaryregions further comprise a self complementary, palindrome, or repeatregion. The dashed portions of each polynucleotide sequence of themultifunctional siNA construct have complementarity with regard tocorresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. FIG. 18B shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe first and second complementary regions are situated at the 5′-endsof each polynucleotide sequence in the multifunctional siNA, and whereinthe first and second complementary regions further comprise a selfcomplementary, palindrome, or repeat region. The dashed portions of eachpolynucleotide sequence of the multifunctional siNA construct havecomplementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences.

FIG. 19 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising a single polynucleotide sequence comprisingdistinct regions that are each capable of mediating RNAi directedcleavage of differing target nucleic acid sequences and wherein themultifunctional siNA construct further comprises a self complementary,palindrome, or repeat region, thus enabling shorter bifuctional siNAconstructs that can mediate RNA interference against differing targetnucleic acid sequences. FIG. 19A shows a non-limiting example of amultifunctional siNA molecule having a first region that iscomplementary to a first target nucleic acid sequence (complementaryregion 1) and a second region that is complementary to a second targetnucleic acid sequence (complementary region 2), wherein the secondcomplementary region is situated at the 3′-end of the polynucleotidesequence in the multifunctional siNA, and wherein the first and secondcomplementary regions further comprise a self complementary, palindrome,or repeat region. The dashed portions of each polynucleotide sequence ofthe multifunctional siNA construct have complementarity with regard tocorresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. FIG. 19B shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe first complementary region is situated at the 5′-end of thepolynucleotide sequence in the multifunctional siNA, and wherein thefirst and second complementary regions further comprise a selfcomplementary, palindrome, or repeat region. The dashed portions of eachpolynucleotide sequence of the multifunctional siNA construct havecomplementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. In one embodiment, these multifunctional siNA constructs areprocessed in vivo or in vitro to generate multifunctional siNAconstructs as shown in FIG. 18.

FIG. 20 shows a non-limiting example of how multifunctional siNAmolecules of the invention can target two separate target nucleic acidmolecules, such as separate RNA molecules encoding differing proteins,for example, a cytokine and its corresponding receptor, differing viralstrains, a virus and a cellular protein involved in viral infection orreplication, or differing proteins involved in a common or divergentbiologic pathway that is implicated in the maintenance of progression ofdisease. Each strand of the multifunctional siNA construct comprises aregion having complementarity to separate target nucleic acid molecules.The multifunctional siNA molecule is designed such that each strand ofthe siNA can be utilized by the RISC complex to initiate RNAinterference mediated cleavage of its corresponding target. These designparameters can include destabilization of each end of the siNA construct(see for example Schwarz et al., 2003, Cell, 115, 199-208). Suchdestabilization can be accomplished for example by usingguanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), ordestabilizing chemically modified nucleotides at terminal nucleotidepositions as is known in the art.

FIG. 21 shows a non-limiting example of how multifunctional siNAmolecules of the invention can target two separate target nucleic acidsequences within the same target nucleic acid molecule, such asalternate coding regions of a RNA, coding and non-coding regions of aRNA, or alternate splice variant regions of a RNA. Each strand of themultifunctional siNA construct comprises a region having complementarityto the separate regions of the target nucleic acid molecule. Themultifunctional siNA molecule is designed such that each strand of thesiNA can be utilized by the RISC complex to initiate RNA interferencemediated cleavage of its corresponding target region. These designparameters can include destabilization of each end of the siNA construct(see for example Schwarz et al., 2003, Cell, 115, 199-208). Suchdestabilization can be accomplished for example by usingguanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), ordestabilizing chemically modified nucleotides at terminal nucleotidepositions as is known in the art.

FIG. 22 shows a non-limiting example of reduction of SCD mRNA in A549cells mediated by chemically modified siNAs that target SCD mRNA. A549cells were transfected with 0.25 ug/well of lipid complexed with 25 nMsiNA. Active siNA constructs comprising various stabilizationchemistries (see Tables III and IV) were compared to untreated cells, amatched chemistry irrelevant siNA control construct (IC; 32072/32075),and cells transfected with lipid alone (transfection control). As shownin the figure, the siNA constructs significantly reduce SCD RNAexpression.

DETAILED DESCRIPTION OF THE INVENTION

Mechanism of Action of Nucleic Acid Molecules of the Invention

The discussion that follows discusses the proposed mechanism of RNAinterference mediated by short interfering RNA as is presently known,and is not meant to be limiting and is not an admission of prior art.Applicant demonstrates herein that chemically-modified short interferingnucleic acids possess similar or improved capacity to mediate RNAi as dosiRNA molecules and are expected to possess improved stability andactivity in vivo; therefore, this discussion is not meant to be limitingonly to siRNA and can be applied to siNA as a whole. By “improvedcapacity to mediate RNAi” or “improved RNAi activity” is meant toinclude RNAi activity measured in vitro and/or in vivo where the RNAiactivity is a reflection of both the ability of the siNA to mediate RNAiand the stability of the siNAs of the invention. In this invention, theproduct of these activities can be increased in vitro and/or in vivocompared to an all RNA siRNA or a siNA containing a plurality ofribonucleotides. In some cases, the activity or stability of the siNAmolecule can be decreased (i.e., less than ten-fold), but the overallactivity of the siNA molecule is enhanced in vitro and/or in vivo.

RNA interference refers to the process of sequence specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes whichis commonly shared by diverse flora and phyla (Fire et al., 1999, TrendsGenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or the random integration of transposonelements into a host genome via a cellular response that specificallydestroys homologous single-stranded RNA or viral genomic RNA. Thepresence of dsRNA in cells triggers the RNAi response though a mechanismthat has yet to be fully characterized. This mechanism appears to bedifferent from the interferon response that results from dsRNA-mediatedactivation of protein kinase PKR and 2′, 5′-oligoadenylate synthetaseresulting in non-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as Dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363).Short interfering RNAs derived from Dicer activity are typically about21 to about 23 nucleotides in length and comprise about 19 base pairduplexes.

Dicer has also been implicated in the excision of 21 - and 22-nucleotidesmall temporal RNAs (stRNAs) from precursor RNA of conserved structurethat are implicated in translational control (Hutvagner et al., 2001,Science, 293, 834). The RNAi response also features an endonucleasecomplex containing a siRNA, commonly referred to as an RNA-inducedsilencing complex (RISC), which mediates cleavage of single-stranded RNAhaving sequence homologous to the siRNA. Cleavage of the target RNAtakes place in the middle of the region complementary to the guidesequence of the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15,188). In addition, RNA interference can also involve small RNA (e.g.,micro-RNA or miRNA) mediated gene silencing, presumably though cellularmechanisms that regulate chromatin structure and thereby preventtranscription of target gene sequences (see for example Allshire, 2002,Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837;Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science,297, 2232-2237). As such, siNA molecules of the invention can be used tomediate gene silencing via interaction with RNA transcripts oralternately by interaction with particular gene sequences, wherein suchinteraction results in gene silencing either at the transcriptionallevel or post-transcriptional level.

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans. Wiannyand Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated bydsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describeRNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001,Nature, 411, 494, describe RNAi induced by introduction of duplexes ofsynthetic 21 -nucleotide RNAs in cultured mammalian cells includinghuman embryonic kidney and HeLa cells. Recent work in Drosophilaembryonic lysates has revealed certain requirements for siRNA length,structure, chemical composition, and sequence that are essential tomediate efficient RNAi activity. These studies have shown that 21nucleotide siRNA duplexes are most active when containing two2-nucleotide 3′-terminal nucleotide overhangs. Furthermore, substitutionof one or both siRNA strands with 2′-deoxy or 2′-O-methyl nucleotidesabolishes RNAi activity, whereas substitution of 3′-terminal siRNAnucleotides with deoxy nucleotides was shown to be tolerated. Mismatchsequences in the center of the siRNA duplex were also shown to abolishRNAi activity. In addition, these studies also indicate that theposition of the cleavage site in the target RNA is defined by the 5′-endof the siRNA guide sequence rather than the 3′-end (Elbashir et al.,2001, EMBO J., 20, 6877). Other studies have indicated that a5′-phosphate on the target-complementary strand of a siRNA duplex isrequired for siRNA activity and that ATP is utilized to maintain the5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309);however, siRNA molecules lacking a 5′-phosphate are active whenintroduced exogenously, suggesting that 5′-phosphorylation of siRNAconstructs may occur in vivo.

Synthesis of Nucleic Acid Molecules

Synthesis of nucleic acids greater than 100 nucleotides in length isdifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small nucleic acid motifs(“small” refers to nucleic acid motifs no more than 100 nucleotides inlength, preferably no more than 80 nucleotides in length, and mostpreferably no more than 50 nucleotides in length; e.g., individual siNAoligonucleotide sequences or siNA sequences synthesized in tandem) arepreferably used for exogenous delivery. The simple structure of thesemolecules increases the ability of the nucleic acid to invade targetedregions of protein and/or RNA structure. Exemplary molecules of theinstant invention are chemically synthesized, and others can similarlybe synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example as described in Caruthers etal., 1992, Methods in Enzymology 211, 3-19, Thompson et al.,International PCT Publication No. WO 99/54459, Wincott et al., 1995,Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol.Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, andBrennan, U.S. Pat. No. 6,001,311. All of these references areincorporated herein by reference. The synthesis of oligonucleotidesmakes use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In anon-limiting example, small scale syntheses are conducted on a 394Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocolwith a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45second coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoronucleotides. Table V outlines the amounts and the contact times of thereagents used in the synthesis cycle. Alternatively, syntheses at the0.2 μmol scale can be performed on a 96-well plate synthesizer, such asthe instrument produced by Protogene (Palo Alto, Calif.) with minimalmodification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol)of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyltetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycleof 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-foldexcess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-foldexcess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used ineach coupling cycle of deoxy residues relative to polymer-bound5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc.synthesizer, determined by calorimetric quantitation of the tritylfractions, are typically 97.5-99%. Other oligonucleotide synthesisreagents for the 394 Applied Biosystems, Inc. synthesizer include thefollowing: detritylation solution is 3% TCA in methylene chloride (ABI);capping is performed with 16% N-methyl imidazole in THF (ABI) and 10%acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solutionis 16.9 mM I₂, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems,Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directlyfrom the reagent bottle. S-Ethyltetrazole solution (0.25 M inacetonitrile) is made up from the solid obtained from AmericanInternational Chemical, Inc. Alternately, for the introduction ofphosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one1,1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the DNA-based oligonucleotides is performed as follows:the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mLglass screw top vial and suspended in a solution of 40% aqueousmethylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C.,the supernatant is removed from the polymer support. The support iswashed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and thesupernatant is then added to the first supernatant. The combinedsupernatants, containing the oligoribonucleotide, are dried to a whitepowder.

The method of synthesis used for RNA including certain siNA molecules ofthe invention follows the procedure as described in Usman et al., 1987,J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res.,18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of commonnucleic acid protecting and coupling groups, such as dimethoxytrityl atthe 5′-end, and phosphoramidites at the 3′-end. In a non-limitingexample, small scale syntheses are conducted on a 394 AppliedBiosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5min coupling step for alkylsilyl protected nucleotides and a 2.5 mincoupling step for 2′-O-methylated nucleotides. Table V outlines theamounts and the contact times of the reagents used in the synthesiscycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a96-well plate synthesizer, such as the instrument produced by Protogene(Palo Alto, Calif.) with minimal modification to the cycle. A 33-foldexcess (60 μL of 0.11 M =6.6 μmol) of 2′-O-methyl phosphoramidite and a75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can beused in each coupling cycle of 2′-O-methyl residues relative topolymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol)of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess ofS-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in eachcoupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl.Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer,determined by colorimetric quantitation of the trityl fractions, aretypically 97.5-99%. Other oligonucleotide synthesis reagents for the 394Applied Biosystems, Inc. synthesizer include the following:detritylation solution is 3% TCA in methylene chloride (ABI); capping isperformed with 16% N-methyl imidazole in THF (ABI) and 10% aceticanhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mMI₂, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.).Burdick & Jackson Synthesis Grade acetonitrile is used directly from thereagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) ismade up from the solid obtained from American International Chemical,Inc. Alternately, for the introduction of phosphorothioate linkages,Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide0.05 M inacetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-potprotocol. For the two-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10min. After cooling to −20° C., the supernatant is removed from thepolymer support. The support is washed three times with 1.0 mL ofEtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to thefirst supernatant. The combined supernatants, containing theoligoribonucleotide, are dried to a white powder. The base deprotectedoligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mLTEA•3HF to provide a 1.4 M HF concentration) and heated to 65° C. After1.5 h, the oligomer is quenched with 1.5 M NH₄HCO₃.

Alternatively, for the one-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL)at 65° C. for 15 minutes. The vial is brought to room temperatureTEA•3HF (0.1 mL) is added and the vial is heated at 65° C. for 15minutes. The sample is cooled at −20° C. and then quenched with 1.5 MNH₄HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had beenprewashed with acetonitrile followed by 50 mM TEAA. After washing theloaded cartridge with water, the RNA is detritylated with 0.5% TFA for13 minutes. The cartridge is then washed again with water, saltexchanged with 1 M NaCl and washed with water again. The oligonucleotideis then eluted with 30% acetonitrile.

The average stepwise coupling yields are typically >98% (Wincott et al.,1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in theart will recognize that the scale of synthesis can be adapted to belarger or smaller than the example described above including but notlimited to 96-well format.

Alternatively, the nucleic acid molecules of the present invention canbe synthesized separately and joined together post-synthetically, forexample, by ligation (Moore et al., 1992, Science 256, 9923; Draper etal., International PCT publication No. WO 93/23569; Shabarova et al.,1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides& Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204),or by hybridization following synthesis and/or deprotection.

The siNA molecules of the invention can also be synthesized via a tandemsynthesis methodology as described in Example 1 herein, wherein bothsiNA strands are synthesized as a single contiguous oligonucleotidefragment or strand separated by a cleavable linker which is subsequentlycleaved to provide separate siNA fragments or strands that hybridize andpermit purification of the siNA duplex. The linker can be apolynucleotide linker or a non-nucleotide linker. The tandem synthesisof siNA as described herein can be readily adapted to bothmultiwell/multiplate synthesis platforms such as 96 well or similarlylarger multi-well platforms. The tandem synthesis of siNA as describedherein can also be readily adapted to large scale synthesis platformsemploying batch reactors, synthesis columns and the like.

A siNA molecule can also be assembled from two distinct nucleic acidstrands or fragments wherein one fragment includes the sense region andthe second fragment includes the antisense region of the RNA molecule.

The nucleic acid molecules of the present invention can be modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H(for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al.,1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purifiedby gel electrophoresis using general methods or can be purified by highpressure liquid chromatography (HPLC; see Wincott et al., supra, thetotality of which is hereby incorporated herein by reference) andre-suspended in water.

In another aspect of the invention, siNA molecules of the invention areexpressed from transcription units inserted into DNA or RNA vectors. Therecombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Therecombinant vectors capable of expressing the siNA molecules can bedelivered as described herein, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of siNA molecules.

Optimizing Activity of the Nucleic Acid Molecule of the Invention.

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) can prevent their degradation by serumribonucleases, which can increase their potency (see e.g., Eckstein etal., International Publication No. WO 92/07065; Perrault et al., 1990Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman andCedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al.,International Publication No. WO 93/15187; and Rossi et al.,International Publication No. WO 91/03162; Sproat, U.S. Pat. No.5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al.,supra; all of which are incorporated by reference herein). All of theabove references describe various chemical modifications that can bemade to the base, phosphate and/or sugar moieties of the nucleic acidmolecules described herein. Modifications that enhance their efficacy incells, and removal of bases from nucleic acid molecules to shortenoligonucleotide synthesis times and reduce chemical requirements aredesired.

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for areview see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994,Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35,14090). Sugar modification of nucleic acid molecules have beenextensively described in the art (see Eckstein et al., InternationalPublication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344,565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren,Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. InternationalPublication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 andBeigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al.,International PCT publication No. WO 97/26270; Beigelman et al., U.S.Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al.,International PCT Publication No. WO 98/13526; Thompson et al., U.S.Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al.,1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers(Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev.Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5,1999-2010; all of the references are hereby incorporated in theirtotality by reference herein). Such publications describe generalmethods and strategies to determine the location of incorporation ofsugar, base and/or phosphate modifications and the like into nucleicacid molecules without modulating catalysis, and are incorporated byreference herein. In view of such teachings, similar modifications canbe used as described herein to modify the siNA nucleic acid molecules ofthe instant invention so long as the ability of siNA to promote RNAi iscells is not significantly inhibited.

While chemical modification of oligonucleotide internucleotide linkageswith phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonatelinkages improves stability, excessive modifications can cause sometoxicity or decreased activity. Therefore, when designing nucleic acidmolecules, the amount of these internucleotide linkages should beminimized. The reduction in the concentration of these linkages shouldlower toxicity, resulting in increased efficacy and higher specificityof these molecules.

Short interfering nucleic acid (siNA) molecules having chemicalmodifications that maintain or enhance activity are provided. Such anucleic acid is also generally more resistant to nucleases than anunmodified nucleic acid. Accordingly, the in vitro and/or in vivoactivity should not be significantly lowered. In cases in whichmodulation is the goal, therapeutic nucleic acid molecules deliveredexogenously should optimally be stable within cells until translation ofthe target RNA has been modulated long enough to reduce the levels ofthe undesirable protein. This period of time varies between hours todays depending upon the disease state. Improvements in the chemicalsynthesis of RNA and DNA (Wincott et al., 1995, Nucleic Acids Res. 23,2677; Caruthers et al., 1992, Methods in Enzymology 211, 3-19(incorporated by reference herein)) have expanded the ability to modifynucleic acid molecules by introducing nucleotide modifications toenhance their nuclease stability, as described above.

In one embodiment, nucleic acid molecules of the invention include oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clampnucleotides. A G-clamp nucleotide is a modified cytosine analog whereinthe modifications confer the ability to hydrogen bond both Watson-Crickand Hoogsteen faces of a complementary guanine within a duplex, see forexample Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. Asingle G-clamp analog substitution within an oligonucleotide can resultin substantially enhanced helical thermal stability and mismatchdiscrimination when hybridized to complementary oligonucleotides. Theinclusion of such nucleotides in nucleic acid molecules of the inventionresults in both enhanced affinity and specificity to nucleic acidtargets, complementary sequences, or template strands. In anotherembodiment, nucleic acid molecules of the invention include one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleicacid” nucleotides such as a 2′, 4′-C methylene bicyclo nucleotide (seefor example Wengel et al., International PCT Publication No. WO 00/66604and WO 99/14226).

In another embodiment, the invention features conjugates and/orcomplexes of siNA molecules of the invention. Such conjugates and/orcomplexes can be used to facilitate delivery of siNA molecules into abiological system, such as a cell. The conjugates and complexes providedby the instant invention can impart therapeutic activity by transferringtherapeutic compounds across cellular membranes, altering thepharmacokinetics, and/or modulating the localization of nucleic acidmolecules of the invention. The present invention encompasses the designand synthesis of novel conjugates and complexes for the delivery ofmolecules, including, but not limited to, small molecules, lipids,cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids,antibodies, toxins, negatively charged polymers and other polymers, forexample proteins, peptides, hormones, carbohydrates, polyethyleneglycols, or polyamines, across cellular membranes. In general, thetransporters described are designed to be used either individually or aspart of a multi-component system, with or without degradable linkers.These compounds are expected to improve delivery and/or localization ofnucleic acid molecules of the invention into a number of cell typesoriginating from different tissues, in the presence or absence of serum(see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of themolecules described herein can be attached to biologically activemolecules via linkers that are biodegradable, such as biodegradablenucleic acid linker molecules.

The term “biodegradable linker” as used herein, refers to a nucleic acidor non-nucleic acid linker molecule that is designed as a biodegradablelinker to connect one molecule to another molecule, for example, abiologically active molecule to a siNA molecule of the invention or thesense and antisense strands of a siNA molecule of the invention. Thebiodegradable linker is designed such that its stability can bemodulated for a particular purpose, such as delivery to a particulartissue or cell type. The stability of a nucleic acid-based biodegradablelinker molecule can be modulated by using various chemistries, forexample combinations of ribonucleotides, deoxyribonucleotides, andchemically-modified nucleotides, such as 2′-O-methyl, 2′-fluoro,2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified orbase modified nucleotides. The biodegradable nucleic acid linkermolecule can be a dimer, trimer, tetramer or longer nucleic acidmolecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length,or can comprise a single nucleotide with a phosphorus-based linkage, forexample, a phosphoramidate or phosphodiester linkage. The biodegradablenucleic acid linker molecule can also comprise nucleic acid backbone,nucleic acid sugar, or nucleic acid base modifications.

The term “biodegradable” as used herein, refers to degradation in abiological system, for example, enzymatic degradation or chemicaldegradation.

The term “biologically active molecule” as used herein refers tocompounds or molecules that are capable of eliciting or modifying abiological response in a system. Non-limiting examples of biologicallyactive siNA molecules either alone or in combination with othermolecules contemplated by the instant invention include therapeuticallyactive molecules such as antibodies, cholesterol, hormones, antivirals,peptides, proteins, chemotherapeutics, small molecules, vitamins,co-factors, nucleosides, nucleotides, oligonucleotides, enzymaticnucleic acids, antisense nucleic acids, triplex formingoligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers,decoys and analogs thereof. Biologically active molecules of theinvention also include molecules capable of modulating thepharmacokinetics and/or pharmacodynamics of other biologically activemolecules, for example, lipids and polymers such as polyamines,polyamides, polyethylene glycol and other polyethers.

The term “phospholipid” as used herein, refers to a hydrophobic moleculecomprising at least one phosphorus group. For example, a phospholipidcan comprise a phosphorus-containing group and saturated or unsaturatedalkyl group, optionally substituted with OH, COOH, oxo, amine, orsubstituted or unsubstituted aryl groups.

Therapeutic nucleic acid molecules (e.g., siNA molecules) deliveredexogenously optimally are stable within cells until reversetranscription of the RNA has been modulated long enough to reduce thelevels of the RNA transcript. The nucleic acid molecules are resistantto nucleases in order to function as effective intracellular therapeuticagents. Improvements in the chemical synthesis of nucleic acid moleculesdescribed in the instant invention and in the art have expanded theability to modify nucleic acid molecules by introducing nucleotidemodifications to enhance their nuclease stability as described above.

In yet another embodiment, siNA molecules having chemical modificationsthat maintain or enhance enzymatic activity of proteins involved in RNAiare provided. Such nucleic acids are also generally more resistant tonucleases than unmodified nucleic acids. Thus, in vitro and/or in vivothe activity should not be significantly lowered.

Use of the nucleic acid-based molecules of the invention will lead tobetter treatments by affording the possibility of combination therapies(e.g., multiple siNA molecules targeted to different genes; nucleic acidmolecules coupled with known small molecule modulators; or intermittenttreatment with combinations of molecules, including different motifsand/or other chemical or biological molecules). The treatment ofsubjects with siNA molecules can also include combinations of differenttypes of nucleic acid molecules, such as enzymatic nucleic acidmolecules (ribozymes), allozymes, antisense, 2,5-A oligoadenylate,decoys, and aptamers.

In another aspect a siNA molecule of the invention comprises one or more5′ and/or a 3′-cap structure, for example, on only the sense siNAstrand, the antisense siNA strand, or both siNA strands.

By “cap structure” is meant chemical modifications, which have beenincorporated at either terminus of the oligonucleotide (see, forexample, Adamic et al., U.S. Pat. No. 5,998,203, incorporated byreference herein). These terminal modifications protect the nucleic acidmolecule from exonuclease degradation, and may help in delivery and/orlocalization within a cell. The cap may be present at the 5′-terminus(5′-cap) or at the 3′-terminal (3′-cap) or may be present on bothtermini. In non-limiting examples, the 5′-cap includes, but is notlimited to, glyceryl, inverted deoxy abasic residue (moiety);4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide,4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitolnucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide;phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety;3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety;3′-2′-inverted abasic moiety; 1,4-butanediol phosphate;3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate;3′-phosphorothioate; phosphorodithioate; or bridging or non-bridgingmethylphosphonate moiety. Non-limiting examples of cap moieties areshown in FIG. 10.

Non-limiting examples of the 3′-cap include, but are not limited to,glyceryl, inverted deoxy abasic residue (moiety), 4′, 5′-methylenenucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide,carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propylphosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate;1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitolnucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

By the term “non-nucleotide” is meant any group or compound which can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound is abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymine and therefore lacks a base at the 1′-position.

An “alkyl” group refers to a saturated aliphatic hydrocarbon, includingstraight-chain, branched-chain, and cyclic alkyl groups. Preferably, thealkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl offrom 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group canbe substituted or unsubstituted. When substituted the substitutedgroup(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂,amino, or SH. The term also includes alkenyl groups that are unsaturatedhydrocarbon groups containing at least one carbon-carbon double bond,including straight-chain, branched-chain, and cyclic groups. Preferably,the alkenyl group has 1 to 12 carbons. More preferably, it is a loweralkenyl of from 1 to 7 carbons, more preferably I to 4 carbons. Thealkenyl group may be substituted or unsubstituted. When substituted thesubstituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S,NO₂, halogen, N(CH₃)₂, amino, or SH. The term “alkyl” also includesalkynyl groups that have an unsaturated hydrocarbon group containing atleast one carbon-carbon triple bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkynyl group has 1to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7carbons, more preferably 1 to 4 carbons. The alkynyl group may besubstituted or unsubstituted. When substituted the substituted group(s)is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂, amino orSH.

Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl,heterocyclic aryl, amide and ester groups. An “aryl” group refers to anaromatic group that has at least one ring having a conjugated pielectron system and includes carbocyclic aryl, heterocyclic aryl andbiaryl groups, all of which may be optionally substituted. The preferredsubstituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH,OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An“alkylaryl” group refers to an alkyl group (as described above)covalently joined to an aryl group (as described above). Carbocyclicaryl groups are groups wherein the ring atoms on the aromatic ring areall carbon atoms. The carbon atoms are optionally substituted.Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms asring atoms in the aromatic ring and the remainder of the ring atoms arecarbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen,and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo,pyrimidyl, pyrazinyl, imidazolyl and the like, all optionallysubstituted. An “amide” refers to an —C(O)—NH—R, where R is eitheralkyl, aryl, alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′,where R is either alkyl, aryl, alkylaryl or hydrogen.

By “nucleotide” as used herein is as recognized in the art to includenatural bases (standard), and modified bases well known in the art. Suchbases are generally located at the 1′ position of a nucleotide sugarmoiety. Nucleotides generally comprise a base, sugar and a phosphategroup. The nucleotides can be unmodified or modified at the sugar,phosphate and/or base moiety, (also referred to interchangeably asnucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides and other; see, for example, Usman andMcSwiggen, supra; Eckstein et al., International PCT Publication No. WO92/07065; Usman et al., International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra, all are hereby incorporated by referenceherein). There are several examples of modified nucleic acid bases knownin the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22,2183. Some of the non-limiting examples of base modifications that canbe introduced into nucleic acid molecules include, inosine, purine,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidinesor 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others(Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine and uracil at 1′position or theirequivalents.

In one embodiment, the invention features modified siNA molecules, withphosphate backbone modifications comprising one or morephosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and/or alkylsilyl, substitutions. For a review ofoligonucleotide backbone modifications, see Hunziker and Leumann, 1995,Nucleic Acid Analogues: Synthesis and Properties, in Modern SyntheticMethods, VCH, 331-417, and Mesmaeker et al., 1994, Novel BackboneReplacements for Oligonucleotides, in Carbohydrate Modifications inAntisense Research, ACS, 24-39.

By “abasic” is meant sugar moieties lacking a base or having otherchemical groups in place of a base at the 1′ position, see for exampleAdamic et al., U.S. Pat. No. 5,998,203.

By “unmodified nucleoside” is meant one of the bases adenine, cytosine,guanine, thymine, or uracil joined to the 1′ carbon ofP-D-ribo-furanose.

By “modified nucleoside” is meant any nucleotide base which contains amodification in the chemical structure of an unmodified nucleotide base,sugar and/or phosphate. Non-limiting examples of modified nucleotidesare shown by Formulae I-VII and/or other modifications described herein.

In connection with 2′-modified nucleotides as described for the presentinvention, by “amino” is meant 2′-NH2 or 2′-O- NH2, which can bemodified or unmodified. Such modified groups are described, for example,in Eckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al.,U.S. Pat. No. 6,248,878, which are both incorporated by reference intheir entireties.

Various modifications to nucleic acid siNA structure can be made toenhance the utility of these molecules. Such modifications will enhanceshelf-life, half-life in vitro, stability, and ease of introduction ofsuch oligonucleotides to the target site, e.g., to enhance penetrationof cellular membranes, and confer the ability to recognize and bind totargeted cells.

Administration of Nucleic Acid Molecules

A siNA molecule of the invention can be adapted for use to prevent ortreat diabetes (type I and/or type II), atherosclerosis, cancer,obesity, and viral infection, and/or any other trait, disease, disorderor condition that is related to or will respond to the levels of SCD ina cell or tissue, alone or in combination with other therapies. Forexample, a siNA molecule can comprise a delivery vehicle, includingliposomes, for administration to a subject, carriers and diluents andtheir salts, and/or can be present in pharmaceutically acceptableformulations. Methods for the delivery of nucleic acid molecules aredescribed in Akhtar et al., 1992, Trends Cell Bio., 2, 139; DeliveryStrategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995,Maurer et al., 1999, Mol. Membr. Biol., 16, 129-140; Hofland and Huang,1999, Handb. Exp. Pharmacol., 137, 165-192; and Lee et al., 2000, ACSSymp. Ser., 752, 184-192, all of which are incorporated herein byreference. Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan etal., PCT WO 94/02595 further describe the general methods for deliveryof nucleic acid molecules. These protocols can be utilized for thedelivery of virtually any nucleic acid molecule. Nucleic acid moleculescan be administered to cells by a variety of methods known to those ofskill in the art, including, but not restricted to, encapsulation inliposomes, by iontophoresis, or by incorporation into other vehicles,such as biodegradable polymers, hydrogels, cyclodextrins (see forexample Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wanget al., International PCT publication Nos. WO 03/47518 and WO 03/46185),poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see forexample U.S. Pat. No. 6,447,796 and U.S. patent application PublicationNo. U.S. 2002130430), biodegradable nanocapsules, and bioadhesivemicrospheres, or by proteinaceous vectors (O'Hare and Normand,International PCT Publication No. WO 00/53722). In another embodiment,the nucleic acid molecules of the invention can also be formulated orcomplexed with polyethyleneimine and derivatives thereof, such aspolyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL)or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine(PEI-PEG-triGAL) derivatives. In one embodiment, the nucleic acidmolecules of the invention are formulated as described in U.S. patentapplication Publication No. 20030077829, incorporated by referenceherein in its entirety.

In one embodiment, a siNA molecule of the invention is complexed withmembrane disruptive agents such as those described in U.S. patentapplication Publication No. 20010007666, incorporated by referenceherein in its entirety including the drawings. In another embodiment,the membrane disruptive agent or agents and the siNA molecule are alsocomplexed with a cationic lipid or helper lipid molecule, such as thoselipids described in U.S. Pat. No. 6,235,310, incorporated by referenceherein in its entirety including the drawings.

In one embodiment, a siNA molecule of the invention is complexed withdelivery systems as described in U.S. patent application Publication No.2003077829 and International PCT Publication Nos. WO 00/03683 and WO02/087541, all incorporated by reference herein in their entiretyincluding the drawings.

In one embodiment, the nucleic acid molecules of the invention areadministered via pulmonary delivery, such as by inhalation of an aerosolor spray dried formulation administered by an inhalation device ornebulizer, providing rapid local uptake of the nucleic acid moleculesinto relevant pulmonary tissues. Solid particulate compositionscontaining respirable dry particles of micronized nucleic acidcompositions can be prepared by grinding dried or lyophilized nucleicacid compositions, and then passing the micronized composition through,for example, a 400 mesh screen to break up or separate out largeagglomerates. A solid particulate composition comprising the nucleicacid compositions of the invention can optionally contain a dispersantwhich serves to facilitate the formation of an aerosol as well as othertherapeutic compounds. A suitable dispersant is lactose, which can beblended with the nucleic acid compound in any suitable ratio, such as a1 to 1 ratio by weight.

Aerosols of liquid particles comprising a nucleic acid composition ofthe invention can be produced by any suitable means, such as with anebulizer (see for example U.S. Pat. No. 4,501,729). Nebulizers arecommercially available devices which transform solutions or suspensionsof an active ingredient into a therapeutic aerosol mist either by meansof acceleration of a compressed gas, typically air or oxygen, through anarrow venturi orifice or by means of ultrasonic agitation. Suitableformulations for use in nebulizers comprise the active ingredient in aliquid carrier in an amount of up to 40% w/w preferably less than 20%w/w of the formulation. The carrier is typically water or a diluteaqueous alcoholic solution, preferably made isotonic with body fluids bythe addition of, for example, sodium chloride or other suitable salts.Optional additives include preservatives if the formulation is notprepared sterile, for example, methyl hydroxybenzoate, anti-oxidants,flavorings, volatile oils, buffering agents and emulsifiers and otherformulation surfactants. The aerosols of solid particles comprising theactive composition and surfactant can likewise be produced with anysolid particulate aerosol generator. Aerosol generators foradministering solid particulate therapeutics to a subject produceparticles which are respirable, as explained above, and generate avolume of aerosol containing a predetermined metered dose of atherapeutic composition at a rate suitable for human administration. Oneillustrative type of solid particulate aerosol generator is aninsufflator. Suitable formulations for administration by insufflationinclude finely comminuted powders which can be delivered by means of aninsufflator. In the insufflator, the powder, e.g., a metered dosethereof effective to carry out the treatments described herein, iscontained in capsules or cartridges, typically made of gelatin orplastic, which are either pierced or opened in situ and the powderdelivered by air drawn through the device upon inhalation or by means ofa manually-operated pump. The powder employed in the insufflatorconsists either solely of the active ingredient or of a powder blendcomprising the active ingredient, a suitable powder diluent, such aslactose, and an optional surfactant. The active ingredient typicallycomprises from 0.1 to 100 w/w of the formulation. A second type ofillustrative aerosol generator comprises a metered dose inhaler. Metereddose inhalers are pressurized aerosol dispensers, typically containing asuspension or solution formulation of the active ingredient in aliquified propellant. During use these devices discharge the formulationthrough a valve adapted to deliver a metered volume to produce a fineparticle spray containing the active ingredient. Suitable propellantsinclude certain chlorofluorocarbon compounds, for example,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane and mixtures thereof. The formulation canadditionally contain one or more co-solvents, for example, ethanol,emulsifiers and other formulation surfactants, such as oleic acid orsorbitan trioleate, anti-oxidants and suitable flavoring agents. Othermethods for pulmonary delivery are described in, for example U.S. patentapplication Ser. No. 20040037780, and U.S. Pat. Nos. 6,592,904;6,582,728; 6,565,885.

In one embodiment, delivery systems of the invention include, forexample, aqueous and nonaqueous gels, creams, multiple emulsions,microemulsions, liposomes, ointments, aqueous and nonaqueous solutions,lotions, aerosols, hydrocarbon bases and powders, and can containexcipients such as solubilizers, permeation enhancers (e.g., fattyacids, fatty acid esters, fatty alcohols and amino acids), andhydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). Inone embodiment, the pharmaceutically acceptable carrier is a liposome ora transdermal enhancer. Examples of liposomes which can be used in thisinvention include the following: (1) CellFectin, 1:1.5 (M/M) liposomeformulation of the cationic lipidN,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine anddioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); (2) CytofectinGSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (GlenResearch); (3) DOTAP(N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate)(Boehringer Manheim); and (4) Lipofectamine, 3:1 (M/M) liposomeformulation of the polycationic lipid DOSPA and the neutral lipid DOPE(GIBCO BRL).

In one embodiment, delivery systems of the invention include patches,tablets, suppositories, pessaries, gels and creams, and can containexcipients such as solubilizers and enhancers (e.g., propylene glycol,bile salts and amino acids), and other vehicles (e.g., polyethyleneglycol, fatty acid esters and derivatives, and hydrophilic polymers suchas hydroxypropylmethylcellulose and hyaluronic acid).

In one embodiment, siNA molecules of the invention are formulated orcomplexed with polyethylenimine (e.g., linear or branched PEI) and/orpolyethylenimine derivatives, including for example grafted PEIs such asgalactose PEI, cholesterol PEI, antibody derivatized PEI, andpolyethylene glycol PEI (PEG-PEI) derivatives thereof (see for exampleOgris et al., 2001, AAPA PharmSci, 3, 1-11; Furgeson et al., 2003,Bioconjugate Chem., 14, 840-847; Kunath et al., 2002, PhramaceuticalResearch, 19, 810-817; Choi et al., 2001, Bull. Korean Chem. Soc., 22,46-52; Bettinger et al., 1999, Bioconjugate Chem., 10, 558-561; Petersonet al., 2002, Bioconjugate Chem., 13, 845-854; Erbacher et al., 1999,Journal of Gene Medicine Preprint, 1, 1-18; Godbey et al., 1999, PNASUSA, 96, 5177-5181; Godbey et al., 1999, Journal of Controlled Release,60, 149-160; Diebold et al., 1999, Journal of Biological Chemistry, 274,19087-19094; Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645; andSagara, U.S. Pat. No. 6,586,524, incorporated by reference herein.

In one embodiment, a siNA molecule of the invention comprises abioconjugate, for example a nucleic acid conjugate as described inVargeese et al., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003; U.S.Pat. No. 6,528,631; U.S. Pat. No. 6,335,434; U.S. Pat. No. 6,235,886;U.S. Pat. No. 6,153,737; U.S. Pat. No. 5,214,136; U.S. Pat. No.5,138,045, all incorporated by reference herein.

Thus, the invention features a pharmaceutical composition comprising oneor more nucleic acid(s) of the invention in an acceptable carrier, suchas a stabilizer, buffer, and the like. The polynucleotides of theinvention can be administered (e.g., RNA, DNA or protein) and introducedto a subject by any standard means, with or without stabilizers,buffers, and the like, to form a pharmaceutical composition. When it isdesired to use a liposome delivery mechanism, standard protocols forformation of liposomes can be followed. The compositions of the presentinvention can also be formulated and used as creams, gels, sprays, oilsand other suitable compositions for topical, dermal, or transdermaladministration as is known in the art.

The present invention also includes pharmaceutically acceptableformulations of the compounds described. These formulations includesalts of the above compounds, e.g., acid addition salts, for example,salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonicacid.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, e.g., systemic orlocal administration, into a cell or subject, including for example ahuman. Suitable forms, in part, depend upon the use or the route ofentry, for example oral, transdermal, or by injection. Such forms shouldnot prevent the composition or formulation from reaching a target cell(i.e., a cell to which the negatively charged nucleic acid is desirablefor delivery). For example, pharmacological compositions injected intothe blood stream should be soluble. Other factors are known in the art,and include considerations such as toxicity and forms that prevent thecomposition or formulation from exerting its effect.

In one embodiment, siNA molecules of the invention are administered to asubject by systemic administration in a pharmaceutically acceptablecomposition or formulation. By “systemic administration” is meant invivo systemic absorption or accumulation of drugs in the blood streamfollowed by distribution throughout the entire body. Administrationroutes that lead to systemic absorption include, without limitation:intravenous, subcutaneous, intraperitoneal, inhalation, oral,intrapulmonary and intramuscular. Each of these administration routesexposes the siNA molecules of the invention to an accessible diseasedtissue. The rate of entry of a drug into the circulation has been shownto be a function of molecular weight or size. The use of a liposome orother drug carrier comprising the compounds of the instant invention canpotentially localize the drug, for example, in certain tissue types,such as the tissues of the reticular endothelial system (RES). Aliposome formulation that can facilitate the association of drug withthe surface of cells, such as, lymphocytes and macrophages is alsouseful. This approach can provide enhanced delivery of the drug totarget cells by taking advantage of the specificity of macrophage andlymphocyte immune recognition of abnormal cells.

By “pharmaceutically acceptable formulation” or “pharmaceuticallyacceptable composition” is meant, a composition or formulation thatallows for the effective distribution of the nucleic acid molecules ofthe instant invention in the physical location most suitable for theirdesired activity. Non-limiting examples of agents suitable forformulation with the nucleic acid molecules of the instant inventioninclude: P-glycoprotein inhibitors (such as Pluronic P85); biodegradablepolymers, such as poly (DL-lactide-coglycolide) microspheres forsustained release delivery (Emerich, DF et al, 1999, Cell Transplant, 8,47-58); and loaded nanoparticles, such as those made ofpolybutylcyanoacrylate. Other non-limiting examples of deliverystrategies for the nucleic acid molecules of the instant inventioninclude material described in Boado et al., 1998, J. Pharm. Sci., 87,1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge etal., 1995, PNAS USA, 92, 5592-5596; Boado, 1995, Adv. Drug DeliveryRev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26,4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

The invention also features the use of the composition comprisingsurface-modified liposomes containing poly (ethylene glycol) lipids(PEG-modified, or long-circulating liposomes or stealth liposomes).These formulations offer a method for increasing the accumulation ofdrugs in target tissues. This class of drug carriers resistsopsonization and elimination by the mononuclear phagocytic system (MPSor RES), thereby enabling longer blood circulation times and enhancedtissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995,95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011).Such liposomes have been shown to accumulate selectively in tumors,presumably by extravasation and capture in the neovascularized targettissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al.,1995,Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomesenhance the pharmacokinetics and pharmacodynamics of DNA and RNA,particularly compared to conventional cationic liposomes which are knownto accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995,42, 24864-24870; Choi et al., International PCT Publication No. WO96/10391; Ansell et al., International PCT Publication No. WO 96/10390;Holland et al., International PCT Publication No. WO 96/10392).Long-circulating liposomes are also likely to protect drugs fromnuclease degradation to a greater extent compared to cationic liposomes,based on their ability to avoid accumulation in metabolically aggressiveMPS tissues such as the liver and spleen.

The present invention also includes compositions prepared for storage oradministration that include a pharmaceutically effective amount of thedesired compounds in a pharmaceutically acceptable carrier or diluent.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985),hereby incorporated by reference herein. For example, preservatives,stabilizers, dyes and flavoring agents can be provided. These includesodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Inaddition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors that those skilled in the medical artswill recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kgbody weight/day of active ingredients is administered dependent uponpotency of the negatively charged polymer.

The nucleic acid molecules of the invention and formulations thereof canbe administered orally, topically, parenterally, by inhalation or spray,or rectally in dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants and/orvehicles. The term parenteral as used herein includes percutaneous,subcutaneous, intravascular (e.g., intravenous), intramuscular, orintrathecal injection or infusion techniques and the like. In addition,there is provided a pharmaceutical formulation comprising a nucleic acidmolecule of the invention and a pharmaceutically acceptable carrier. Oneor more nucleic acid molecules of the invention can be present inassociation with one or more non-toxic pharmaceutically acceptablecarriers and/or diluents and/or adjuvants, and if desired other activeingredients. The pharmaceutical compositions containing nucleic acidmolecules of the invention can be in a form suitable for oral use, forexample, as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsion, hard or soft capsules, orsyrups or elixirs.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents; such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acidDispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilcan be employed including synthetic mono-or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The nucleic acid molecules of the invention can also be administered inthe form of suppositories, e.g., for rectal administration of the drug.These compositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterallyin a sterile medium. The drug, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per subject perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient.

It is understood that the specific dose level for any particular subjectdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

The nucleic acid molecules of the present invention can also beadministered to a subject in combination with other therapeuticcompounds to increase the overall therapeutic effect. The use ofmultiple compounds to treat an indication can increase the beneficialeffects while reducing the presence of side effects.

In one embodiment, the invention comprises compositions suitable foradministering nucleic acid molecules of the invention to specific celltypes. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu,1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes and bindsbranched galactose-terminal glycoproteins, such as asialoorosomucoid(ASOR). In another example, the folate receptor is overexpressed in manycancer cells. Binding of such glycoproteins, synthetic glycoconjugates,or folates to the receptor takes place with an affinity that stronglydepends on the degree of branching of the oligosaccharide chain, forexample, triatennary structures are bound with greater affinity thanbiatenarry or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22,611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee andLee, 1987, Glycoconjugate J., 4, 317-328, obtained this high specificitythrough the use of N-acetyl-D-galactosamine as the carbohydrate moiety,which has higher affinity for the receptor, compared to galactose. This“clustering effect” has also been described for the binding and uptakeof mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom etal., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose,galactosamine, or folate based conjugates to transport exogenouscompounds across cell membranes can provide a targeted delivery approachto, for example, the treatment of liver disease, cancers of the liver,or other cancers. The use of bioconjugates can also provide a reductionin the required dose of therapeutic compounds required for treatment.Furthermore, therapeutic bioavailability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use of nucleicacid bioconjugates of the invention. Non-limiting examples of suchbioconjugates are described in Vargeese et al., U.S. Ser. No.10/201,394, filed Aug. 13, 2001; and Matulic-Adamic et al., U.S. Ser.No. 60/362,016, filed Mar. 6, 2002.

Alternatively, certain siNA molecules of the instant invention can beexpressed within cells from eukaryotic promoters (e.g., Izant andWeintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc.Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad.Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev.,2, 3-15; Dropulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe etal., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad.Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20,4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al.,1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4,45. Those skilled in the art realize that any nucleic acid can beexpressed in eukaryotic cells from the appropriate DNA/RNA vector. Theactivity of such nucleic acids can be augmented by their release fromthe primary transcript by a enzymatic nucleic acid (Draper et al., PCTWO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992,Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic AcidsRes., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21,3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856.

In another aspect of the invention, RNA molecules of the presentinvention can be expressed from transcription units (see for exampleCouture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors.The recombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Inanother embodiment, pol III based constructs are used to express nucleicacid molecules of the invention (see for example Thompson, U.S. Pat.Nos. 5,902,880 and 6,146,886). The recombinant vectors capable ofexpressing the siNA molecules can be delivered as described above, andpersist in target cells. Alternatively, viral vectors can be used thatprovide for transient expression of nucleic acid molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siNAmolecule interacts with the target mRNA and generates an RNAi response.Delivery of siNA molecule expressing vectors can be systemic, such as byintravenous or intra-muscular administration, by administration totarget cells ex-planted from a subject followed by reintroduction intothe subject, or by any other means that would allow for introductioninto the desired target cell (for a review see Couture et al., 1996,TIG., 12, 510).

In one aspect the invention features an expression vector comprising anucleic acid sequence encoding at least one siNA molecule of the instantinvention. The expression vector can encode one or both strands of asiNA duplex, or a single self-complementary strand that self hybridizesinto a siNA duplex. The nucleic acid sequences encoding the siNAmolecules of the instant invention can be operably linked in a mannerthat allows expression of the siNA molecule (see for example Paul etal., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002,Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology,19, 500; and Novina et al., 2002, Nature Medicine, advance onlinepublication doi:10.1038/nm725).

In another aspect, the invention features an expression vectorcomprising: a) a transcription initiation region (e.g., eukaryotic polI, II or III initiation region); b) a transcription termination region(e.g., eukaryotic pol I, II or III termination region); and c) a nucleicacid sequence encoding at least one of the siNA molecules of the instantinvention, wherein said sequence is operably linked to said initiationregion and said termination region in a manner that allows expressionand/or delivery of the siNA molecule. The vector can optionally includean open reading frame (ORF) for a protein operably linked on the 5′ sideor the 3′-side of the sequence encoding the siNA of the invention;and/or an intron (intervening sequences).

Transcription of the siNA molecule sequences can be driven from apromoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (polII), or RNA polymerase III (pol III). Transcripts from pol II or pol IIIpromoters are expressed at high levels in all cells; the levels of agiven pol III promoter in a given cell type depends on the nature of thegene regulatory sequences (enhancers, silencers, etc.) present nearby.Prokaryotic RNA polymerase promoters are also used, providing that theprokaryotic RNA polymerase enzyme is expressed in the appropriate cells(Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gaoand Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993,Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10,4529-37). Several investigators have demonstrated that nucleic acidmolecules expressed from such promoters can function in mammalian cells(e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwanget al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al.,1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad.Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8;Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U.S.A., 90, 8000-4;Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech,1993, Science, 262, 1566). More specifically, transcription units suchas the ones derived from genes encoding U6 small nuclear (snRNA),transfer RNA (tRNA) and adenovirus VA RNA are useful in generating highconcentrations of desired RNA molecules such as siNA in cells (Thompsonet al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al.,1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No.5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al.,International PCT Publication No. WO 96/18736. The above siNAtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated virus vectors), or viral RNA vectors (such asretroviral or alphavirus vectors) (for a review see Couture andStinchcomb, 1996, supra).

In another aspect the invention features an expression vector comprisinga nucleic acid sequence encoding at least one of the siNA molecules ofthe invention in a manner that allows expression of that siNA molecule.The expression vector comprises in one embodiment; a) a transcriptioninitiation region; b) a transcription termination region; and c) anucleic acid sequence encoding at least one strand of the siNA molecule,wherein the sequence is operably linked to the initiation region and thetermination region in a manner that allows expression and/or delivery ofthe siNA molecule.

In another embodiment the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an open reading frame; and d) a nucleic acid sequence encoding atleast one strand of a siNA molecule, wherein the sequence is operablylinked to the 3′-end of the open reading frame and wherein the sequenceis operably linked to the initiation region, the open reading frame andthe termination region in a manner that allows expression and/ordelivery of the siNA molecule. In yet another embodiment, the expressionvector comprises: a) a transcription initiation region; b) atranscription termination region; c) an intron; and d) a nucleic acidsequence encoding at least one siNA molecule, wherein the sequence isoperably linked to the initiation region, the intron and the terminationregion in a manner which allows expression and/or delivery of thenucleic acid molecule.

In another embodiment, the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an intron; d) an open reading frame; and e) a nucleic acid sequenceencoding at least one strand of a siNA molecule, wherein the sequence isoperably linked to the 3′-end of the open reading frame and wherein thesequence is operably linked to the initiation region, the intron, theopen reading frame and the termination region in a manner which allowsexpression and/or delivery of the siNA molecule.

SCD Biology and Biochemistry

Stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme in thebiosynthesis of monounsaturated fatty acids, including oleate (C18:1)and palmitoleate (C16:1). SCD catalyzes the introduction of the cisdouble bond in the delta-9 position of fatty acyl-CoA substrates, suchas palmitoyl-CoA and stearoyl-CoA, which are converted topalmitoleoyl-CoA (16:1) and oleoyl-CoA (18:1), respectively. These fattyacids are essential components of membrane phospholipids, triglycerides,cholesterol esters, and wax esters. Modulation of phospholipidcomposition ultimately determines physical properties, such as membranefluidity. Further, effects on the composition of cholesterol esters andtriglycerides can substantially affect lipoprotein metabolism andadiposity. SCD expression is sensitive to dietary factors, includingpolyunsaturated fatty acids, cholesterol and vitamin A, hormonal changes(i.e., insulin leptin, and glucagon), developmental processes,temperature changes, thiazolinediones, metals, alcohol, peroxisomalproliferators, and certain phenolic compounds. High SCD activity hasbeen implicated in a wide range of diseases and disorders includingdiabetes, atherosclerosis, cancer, obesity, and viral infection (Ntambiet al., 2002, PNAS USA, 99, 11482-11486).

Several SCD isoforms (SCD1-3) have been identified in the mouse. Micehaving defective SCD expression provide useful information indetermining the relevance of SCD in models of obesity, diabetes, andinsulin resistance. For example, new insights into the physiologicalrole of the SCD1 gene and its endogenous products have been reportedfrom recent studies of the asebia mouse strains (ab^(j) and ab^(2j))that have naturally occurring mutations in SCD1 (Zheng et al., 1999,Nature Genetics, 23, 268-270), as well as a laboratory mouse model witha targeted disruption (SCD1 −/−) (Miyazaki et al., 2001, J. Biol. Chem.,276, 39455-39461). Mice having a targeted disruption of the SCD1 isoformhave reduced body adiposity, increased insulin sensitivity, and areresistant to diet-induced weight gain. The protection from obesity inthese mice involves increased energy expenditure and increased oxygenconsumption. Compared with the wild-type mice, SCD1−/− mice haveincreased levels of plasma ketone bodies, but also have reduced levelsof plasma insulin and leptin. The expression of several genescontrolling lipid oxidation are up-regulated in SCD1−/− mice, whereaslipid synthesis genes are down-regulated in SCD1−/− mice. Theseobservations suggest that a consequence of SCD1 deficiency is anactivation of lipid oxidation in addition to reduced triglyceridesynthesis and storage. Aside from the dramatic alterations intriglyceride and cholesterol metabolism, the SCD1−/− mice areconsiderably leaner than their wild-type counterparts. (Ntambi et al.,2002, PNAS USA, 99, 11482-11486).

The use of small interfering nucleic acid molecules targeting SCD,therefore provides a class of novel therapeutic agents that can be usedin the treatment, alleviation, or prevention of diabetes (type I and/ortype II), atherosclerosis, cancer, obesity, and viral infection, aloneor in combination with other therapies.

EXAMPLES

The following are non-limiting examples showing the selection,isolation, synthesis and activity of nucleic acids of the instantinvention.

Example 1 Tandem Synthesis of siNA Constructs

Exemplary siNA molecules of the invention are synthesized in tandemusing a cleavable linker, for example, a succinyl-based linker. Tandemsynthesis as described herein is followed by a one-step purificationprocess that provides RNAi molecules in high yield. This approach ishighly amenable to siNA synthesis in support of high throughput RNAiscreening, and can be readily adapted to multi-column or multi-wellsynthesis platforms.

After completing a tandem synthesis of a siNA oligo and its complementin which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact(trityl on synthesis), the oligonucleotides are deprotected as describedabove. Following deprotection, the siNA sequence strands are allowed tospontaneously hybridize. This hybridization yields a duplex in which onestrand has retained the 5′-O-DMT group while the complementary strandcomprises a terminal 5′-hydroxyl. The newly formed duplex behaves as asingle molecule during routine solid-phase extraction purification(Trityl-On purification) even though only one molecule has adimethoxytrityl group. Because the strands form a stable duplex, thisdimethoxytrityl group (or an equivalent group, such as other tritylgroups or other hydrophobic moieties) is all that is required to purifythe pair of oligos, for example, by using a C18 cartridge.

Standard phosphoramidite synthesis chemistry is used up to the point ofintroducing a tandem linker, such as an inverted deoxy abasic succinateor glyceryl succinate linker (see FIG. 1) or an equivalent cleavablelinker. A non-limiting example of linker coupling conditions that can beused includes a hindered base such as diisopropylethylamine (DIPA)and/or DMAP in the presence of an activator reagent such asBromotripyrrolidinophosphoniumhexaflurorophosphate (PyBrOP). After thelinker is coupled, standard synthesis chemistry is utilized to completesynthesis of the second sequence leaving the terminal the 5′-O-DMTintact. Following synthesis, the resulting oligonucleotide isdeprotected according to the procedures described herein and quenchedwith a suitable buffer, for example with 50 mM NaOAc or 1.5 M NH₄H₂CO₃.

Purification of the siNA duplex can be readily accomplished using solidphase extraction, for example, using a Waters C18 SepPak 1 g cartridgeconditioned with 1 column volume (CV) of acetonitrile, 2 CV H2O, and 2CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H2O or 50mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with50 mM NaOAc and 50 mM NaCl). The column is then washed, for example with1 CV H2O followed by on-column detritylation, for example by passing 1CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then addinga second CV of 1% aqueous TFA to the column and allowing to stand forapproximately 10 minutes. The remaining TFA solution is removed and thecolumn washed with H2O followed by 1 CV 1M NaCl and additional H2O. ThesiNA duplex product is then eluted, for example, using 1 CV 20% aqueousCAN.

FIG. 2 provides an example of MALDI-TOF mass spectrometry analysis of apurified siNA construct in which each peak corresponds to the calculatedmass of an individual siNA strand of the siNA duplex. The same purifiedsiNA provides three peaks when analyzed by capillary gel electrophoresis(CGE), one peak presumably corresponding to the duplex siNA, and twopeaks presumably corresponding to the separate siNA sequence strands.Ion exchange HPLC analysis of the same siNA contract only shows a singlepeak. Testing of the purified siNA construct using a luciferase reporterassay described below demonstrated the same RNAi activity compared tosiNA constructs generated from separately synthesized oligonucleotidesequence strands.

Example 2 Identification of Potential siNA Target Sites in any RNASequence

The sequence of an RNA target of interest, such as a viral or human mRNAtranscript, is screened for target sites, for example by using acomputer folding algorithm. In a non-limiting example, the sequence of agene or RNA gene transcript derived from a database, such as Genbank, isused to generate siNA targets having complementarity to the target. Suchsequences can be obtained from a database, or can be determinedexperimentally as known in the art. Target sites that are known, forexample, those target sites determined to be effective target sitesbased on studies with other nucleic acid molecules, for exampleribozymes or antisense, or those targets known to be associated with adisease or condition such as those sites containing mutations ordeletions, can be used to design siNA molecules targeting those sites.Various parameters can be used to determine which sites are the mostsuitable target sites within the target RNA sequence. These parametersinclude but are not limited to secondary or tertiary RNA structure, thenucleotide base composition of the target sequence, the degree ofhomology between various regions of the target sequence, or the relativeposition of the target sequence within the RNA transcript. Based onthese determinations, any number of target sites within the RNAtranscript can be chosen to screen siNA molecules for efficacy, forexample by using in vitro RNA cleavage assays, cell culture, or animalmodels. In a non-limiting example, anywhere from 1 to 1000 target sitesare chosen within the transcript based on the size of the siNA constructto be used. High throughput screening assays can be developed forscreening siNA molecules using methods known in the art, such as withmulti-well or multi-plate assays to determine efficient reduction intarget gene expression.

Example 3 Selection of siNA Molecule Target Sites in a RNA

The following non-limiting steps can be used to carry out the selectionof siNAs targeting a given gene sequence or transcript.

-   -   1. The target sequence is parsed in silico into a list of all        fragments or subsequences of a particular length, for example 23        nucleotide fragments, contained within the target sequence. This        step is typically carried out using a custom Perl script, but        commercial sequence analysis programs such as Oligo, MacVector,        or the GCG Wisconsin Package can be employed as well.    -   2. In some instances the siNAs correspond to more than one        target sequence; such would be the case for example in targeting        different transcripts of the same gene, targeting different        transcripts of more than one gene, or for targeting both the        human gene and an animal homolog. In this case, a subsequence        list of a particular length is generated for each of the        targets, and then the lists are compared to find matching        sequences in each list. The subsequences are then ranked        according to the number of target sequences that contain the        given subsequence; the goal is to find subsequences that are        present in most or all of the target sequences. Alternately, the        ranking can identify subsequences that are unique to a target        sequence, such as a mutant target sequence. Such an approach        would enable the use of siNA to target specifically the mutant        sequence and not effect the expression of the normal sequence.    -   3. In some instances the siNA subsequences are absent in one or        more sequences while present in the desired target sequence;        such would be the case if the siNA targets a gene with a        paralogous family member that is to remain untargeted. As in        case 2 above, a subsequence list of a particular length is        generated for each of the targets, and then the lists are        compared to find sequences that are present in the target gene        but are absent in the untargeted paralog.    -   4. The ranked siNA subsequences can be further analyzed and        ranked according to GC content. A preference can be given to        sites containing 30-70% GC, with a further preference to sites        containing 40-60% GC.    -   5. The ranked siNA subsequences can be further analyzed and        ranked according to self-folding and internal hairpins. Weaker        internal folds are preferred; strong hairpin structures are to        be avoided.    -   6. The ranked siNA subsequences can be further analyzed and        ranked according to whether they have runs of GGG or CCC in the        sequence. GGG (or even more Gs) in either strand can make        oligonucleotide synthesis problematic and can potentially        interfere with RNAi activity, so it is avoided whenever better        sequences are available. CCC is searched in the target strand        because that will place GGG in the antisense strand.    -   7. The ranked siNA subsequences can be further analyzed and        ranked according to whether they have the dinucleotide UU        (uridine dinucleotide) on the 3′-end of the sequence, and/or AA        on the 5′-end of the sequence (to yield 3′ WU on the antisense        sequence). These sequences allow one to design siNA molecules        with terminal TT thymidine dinucleotides.    -   8. Four or five target sites are chosen from the ranked list of        subsequences as described above. For example, in subsequences        having 23 nucleotides, the right 21 nucleotides of each chosen        23-mer subsequence are then designed and synthesized for the        upper (sense) strand of the siNA duplex, while the reverse        complement of the left 21 nucleotides of each chosen 23-mer        subsequence are then designed and synthesized for the lower        (antisense) strand of the siNA duplex (see Tables II and III).        If terminal TT residues are desired for the sequence (as        described in paragraph 7), then the two 3′ terminal nucleotides        of both the sense and antisense strands are replaced by TT prior        to synthesizing the oligos.    -   9. The siNA molecules are screened in an in vitro, cell culture        or animal model system to identify the most active siNA molecule        or the most preferred target site within the target RNA        sequence.    -   10. Other design considerations can be used when selecting        target nucleic acid sequences, see, for example, Reynolds et        al., 2004, Nature Biotechnology Advanced Online Publication, 1        Feb. 2004, doi:10.1038/nbt936 and Ui-Tei et al., 2004, Nucleic        Acids Research, 32, doi:10.1093/nar/gkh247.

In an alternate approach, a pool of siNA constructs specific to a SCDtarget sequence is used to screen for target sites in cells expressingSCD RNA, such as such HepG2, MDA-MB-231 or A549 cells. The generalstrategy used in this approach is shown in FIG. 9. A non-limitingexample of such is a pool comprising sequences having any of SEQ ID NOS1-806. Cells expressing SCD are transfected with the pool of siNAconstructs and cells that demonstrate a phenotype associated with SCDinhibition are sorted. The pool of siNA constructs can be expressed fromtranscription cassettes inserted into appropriate vectors (see forexample FIG. 7 and FIG. 8). The siNA from cells demonstrating a positivephenotypic change (e.g., decreased proliferation, decreased SCD mRNAlevels or decreased SCD protein expression), are sequenced to determinethe most suitable target site(s) within the target SCD RNA sequence.

Example 4 SCD Targeted siNA Design

siNA target sites were chosen by analyzing sequences of the SCD RNAtarget and optionally prioritizing the target sites on the basis offolding (structure of any given sequence analyzed to determine siNAaccessibility to the target), by using a library of siNA molecules asdescribed in Example 3, or alternately by using an in vitro siNA systemas described in Example 6 herein. siNA molecules were designed thatcould bind each target and are optionally individually analyzed bycomputer folding to assess whether the siNA molecule can interact withthe target sequence. Varying the length of the siNA molecules can bechosen to optimize activity. Generally, a sufficient number ofcomplementary nucleotide bases are chosen to bind to, or otherwiseinteract with, the target RNA, but the degree of complementarity can bemodulated to accommodate siNA duplexes or varying length or basecomposition. By using such methodologies, siNA molecules can be designedto target sites within any known RNA sequence, for example those RNAsequences corresponding to the any gene transcript.

Chemically modified siNA constructs are designed to provide nucleasestability for systemic administration in vivo and/or improvedpharmacokinetic, localization, and delivery properties while preservingthe ability to mediate RNAi activity. Chemical modifications asdescribed herein are introduced synthetically using synthetic methodsdescribed herein and those generally known in the art. The syntheticsiNA constructs are then assayed for nuclease stability in serum and/orcellular/tissue extracts (e.g. liver extracts). The synthetic siNAconstructs are also tested in parallel for RNAi activity using anappropriate assay, such as a luciferase reporter assay as describedherein or another suitable assay that can quantity RNAi activity.Synthetic siNA constructs that possess both nuclease stability and RNAiactivity can be further modified and re-evaluated in stability andactivity assays. The chemical modifications of the stabilized activesiNA constructs can then be applied to any siNA sequence targeting anychosen RNA and used, for example, in target screening assays to picklead siNA compounds for therapeutic development (see for example FIG.11).

Example 5 Chemical Synthesis and Purification of siNA

siNA molecules can be designed to interact with various sites in the RNAmessage, for example, target sequences within the RNA sequencesdescribed herein. The sequence of one strand of the siNA molecule(s) iscomplementary to the target site sequences described above. The siNAmolecules can be chemically synthesized using methods described herein.Inactive siNA molecules that are used as control sequences can besynthesized by scrambling the sequence of the siNA molecules such thatit is not complementary to the target sequence. Generally, siNAconstructs can by synthesized using solid phase oligonucleotidesynthesis methods as described herein (see for example Usman et al.,U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098;6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos.6,111,086; 6,008,400; 6,111,086 all incorporated by reference herein intheir entirety).

In a non-limiting example, RNA oligonucleotides are synthesized in astepwise fashion using the phosphoramidite chemistry as is known in theart. Standard phosphoramidite chemistry involves the use of nucleosidescomprising any of 5′-O-dimethoxytrityl, 2′-O-tert-butyldimethylsilyl,3′-O-2-Cyanoethyl N,N-diisopropylphosphoroamidite groups, and exocyclicamine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine,and N2-isobutyryl guanosine). Alternately, 2′-O-Silyl Ethers can be usedin conjunction with acid-labile 2′-O-orthoester protecting groups in thesynthesis of RNA as described by Scaringe supra. Differing 2′chemistries can require different protecting groups, for example2′-deoxy-2′-amino nucleosides can utilize N-phthaloyl protection asdescribed by Usman et al., U.S. Pat. No. 5,631,360, incorporated byreference herein in its entirety).

During solid phase synthesis, each nucleotide is added sequentially (3′-to 5′-direction) to the solid support-bound oligonucleotide. The firstnucleoside at the 3′-end of the chain is covalently attached to a solidsupport (e.g., controlled pore glass or polystyrene) using variouslinkers. The nucleotide precursor, a ribonucleoside phosphoramidite, andactivator are combined resulting in the coupling of the secondnucleoside phosphoramidite onto the 5′-end of the first nucleoside. Thesupport is then washed and any unreacted 5′-hydroxyl groups are cappedwith a capping reagent such as acetic anhydride to yield inactive5′-acetyl moieties. The trivalent phosphorus linkage is then oxidized toa more stable phosphate linkage. At the end of the nucleotide additioncycle, the 5′-O-protecting group is cleaved under suitable conditions(e.g., acidic conditions for trityl-based groups and Fluoride forsilyl-based groups). The cycle is repeated for each subsequentnucleotide.

Modification of synthesis conditions can be used to optimize couplingefficiency, for example by using differing coupling times, differingreagent/phosphoramidite concentrations, differing contact times,differing solid supports and solid support linker chemistries dependingon the particular chemical composition of the siNA to be synthesized.Deprotection and purification of the siNA can be performed as isgenerally described in Usman et al., U.S. Pat. No. 5,831,071, U.S. Pat.No. 6,353,098, U.S. Pat. No. 6,437,117, and Bellon et al., U.S. Pat. No.6,054,576, U.S. Pat. No. 6,162,909, U.S. Pat. No. 6,303,773, or Scaringesupra, incorporated by reference herein in their entireties.Additionally, deprotection conditions can be modified to provide thebest possible yield and purity of siNA constructs. For example,applicant has observed that oligonucleotides comprising2′-deoxy-2′-fluoro nucleotides can degrade under inappropriatedeprotection conditions. Such oligonucleotides are deprotected usingaqueous methylamine at about 35° C. for 30 minutes. If the2′-deoxy-2′-fluoro containing oligonucleotide also comprisesribonucleotides, after deprotection with aqueous methylamine at about35° C. for 30 minutes, TEA-HF is added and the reaction maintained atabout 65° C. for an additional 15 minutes.

Example 6 RNAi in vitro Assay to Assess siNA Activity

An in vitro assay that recapitulates RNAi in a cell-free system is usedto evaluate siNA constructs targeting SCD RNA targets. The assaycomprises the system described by Tuschl et al., 1999, Genes andDevelopment, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33adapted for use with SCD target RNA. A Drosophila extract derived fromsyncytial blastoderm is used to reconstitute RNAi activity in vitro.Target RNA is generated via in vitro transcription from an appropriateSCD expressing plasmid using T7 RNA polymerase or via chemical synthesisas described herein. Sense and antisense siNA strands (for example 20 uMeach) are annealed by incubation in buffer (such as 100 mM potassiumacetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 minuteat 90° C. followed by 1 hour at 37° C., then diluted in lysis buffer(for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mMmagnesium acetate). Annealing can be monitored by gel electrophoresis onan agarose gel in TBE buffer and stained with ethidium bromide. TheDrosophila lysate is prepared using zero to two-hour-old embryos fromOregon R flies collected on yeasted molasses agar that are dechorionatedand lysed. The lysate is centrifuged and the supernatant isolated. Theassay comprises a reaction mixture containing 50% lysate [vol/vol], RNA(10-50 pM final concentration), and 10% [vol/vol] lysis buffercontaining siNA (10 nM final concentration). The reaction mixture alsocontains 10 mM creatine phosphate, 10 ug/ml creatine phosphokinase, 100um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin(Promega), and 100 uM of each amino acid. The final concentration ofpotassium acetate is adjusted to 100 mM. The reactions are pre-assembledon ice and preincubated at 25° C. for 10 minutes before adding RNA, thenincubated at 25° C for an additional 60 minutes. Reactions are quenchedwith 4 volumes of 1.25×Passive Lysis Buffer (Promega). Target RNAcleavage is assayed by RT-PCR analysis or other methods known in the artand are compared to control reactions in which siNA is omitted from thereaction.

Alternately, internally-labeled target RNA for the assay is prepared byin vitro transcription in the presence of [alpha-³²P] CTP, passed over aG50 Sephadex column by spin chromatography and used as target RNAwithout further purification. Optionally, target RNA is 5′-³²P-endlabeled using T4 polynucleotide kinase enzyme. Assays are performed asdescribed above and target RNA and the specific RNA cleavage productsgenerated by RNAi are visualized on an autoradiograph of a gel. Thepercentage of cleavage is determined by PHOSPHOR IMAGER®(autoradiography) quantitation of bands representing intact control RNAor RNA from control reactions without siNA and the cleavage productsgenerated by the assay.

In one embodiment, this assay is used to determine target sites in theSCD RNA target for siNA mediated RNAi cleavage, wherein a plurality ofsiNA constructs are screened for RNAi mediated cleavage of the SCD RNAtarget, for example, by analyzing the assay reaction by electrophoresisof labeled target RNA, or by northern blotting, as well as by othermethodology well known in the art.

Example 7 Nucleic Acid Inhibition of SCD Target RNA

siNA molecules targeted to the human SCD RNA are designed andsynthesized as described above. These nucleic acid molecules can betested for cleavage activity in vivo, for example, using the followingprocedure. The target sequences and the nucleotide location within theSCD RNA are given in Tables II and III.

Two formats are used to test the efficacy of siNAs targeting SCD. First,the reagents are tested in cell culture using, for example, HepG2,MDA-MB-231 or A549 cells, to determine the extent of RNA and proteininhibition. siNA reagents (e.g.; see Tables II and III) are selectedagainst the SCD target as described herein. RNA inhibition is measuredafter delivery of these reagents by a suitable transfection agent to,for example, HepG2, MDA-MB-231 or A549 cells. Relative amounts of targetRNA are measured versus actin using real-time PCR monitoring ofamplification (eg., ABI 7700 TAQMAN®). A comparison is made to a mixtureof oligonucleotide sequences made to unrelated targets or to arandomized siNA control with the same overall length and chemistry, butrandomly substituted at each position. Primary and secondary leadreagents are chosen for the target and optimization performed. After anoptimal transfection agent concentration is chosen, a RNA time-course ofinhibition is performed with the lead siNA molecule. In addition, acell-plating format can be used to determine RNA inhibition.

Delivery of siNA to Cells

Cells such as HepG2, MDA-MB-231 or A549 cells are seeded, for example,at 1×10⁵ cells per well of a six-well dish in EGM-2 (BioWhittaker) theday before transfection. siNA (final concentration, for example 20 nM)and cationic lipid (e.g., final concentration 2 μg/ml) are complexed inEGM basal media (Bio Whittaker) at 37° C. for 30 minutes in polystyrenetubes. Following vortexing, the complexed siNA is added to each well andincubated for the times indicated. For initial optimization experiments,cells are seeded, for example, at 1×10³ in 96 well plates and siNAcomplex added as described. Efficiency of delivery of siNA to cells isdetermined using a fluorescent siNA complexed with lipid. Cells in6-well dishes are incubated with siNA for 24 hours, rinsed with PBS andfixed in 2% paraformaldehyde for 15 minutes at room temperature. Uptakeof siNA is visualized using a fluorescent microscope.

TAQMAN® (Real-Time PCR Monitoring of Amplification) and LightcyclerQuantification of mRNA

Total RNA is prepared from cells following siNA delivery, for example,using Qiagen RNA purification kits for 6-well or Rneasy extraction kitsfor 96-well assays. For TAQMAN® analysis (real-time PCR monitoring ofamplification), dual-labeled probes are synthesized with the reporterdye, FAM or JOE, covalently linked at the 5′-end and the quencher dyeTAMRA conjugated to the 3′-end. One-step RT-PCR amplifications areperformed on, for example, an ABI PRISM 7700 Sequence Detector using 50μl reactions consisting of 10 μl total RNA, 100 nM forward primer, 900nM reverse primer, 100 nM probe, 133 TaqMan PCR reaction buffer(PE-Applied Biosystems), 5.5 mM MgCl₂, 300 μM each DATP, dCTP, dGTP, anddTTP, 10U RNase Inhibitor (Promega), 1.25U AMPLITAQ GOLD® (DNApolymerase) (PE-Applied Biosystems) and 10U M-MLV Reverse Transcriptase(Promega). The thermal cycling conditions can consist of 30 minutes at48° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95°C. and 1 minute at 60° C. Quantitation of mRNA levels is determinedrelative to standards generated from serially diluted total cellular RNA(300, 100, 33, 11 ng/rxn) and normalizing to β-actin or GAPDH mRNA inparallel TAQMAN® reactions (real-time PCR monitoring of amplification).For each gene of interest an upper and lower primer and a fluorescentlylabeled probe are designed. Real time incorporation of SYBR Green I dyeinto a specific PCR product can be measured in glass capillary tubesusing a lightcyler. A standard curve is generated for each primer pairusing control cRNA. Values are represented as relative expression toGAPDH in each sample.

Western Blotting

Nuclear extracts can be prepared using a standard micro preparationtechnique (see for example Andrews and Faller, 1991, Nucleic AcidsResearch, 19, 2499). Protein extracts from supernatants are prepared,for example using TCA precipitation. An equal volume of 20% TCA is addedto the cell supernatant, incubated on ice for 1 hour and pelleted bycentrifugation for 5 minutes. Pellets are washed in acetone, dried andresuspended in water. Cellular protein extracts are run on a 10%Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatantextracts) polyacrylamide gel and transferred onto nitro-cellulosemembranes. Non-specific binding can be blocked by incubation, forexample, with 5% non-fat milk for 1 hour followed by primary antibodyfor 16 hour at 4° C. Following washes, the secondary antibody isapplied, for example (1:10,000 dilution) for 1 hour at room temperatureand the signal detected with SuperSignal reagent (Pierce).

Example 8 Models Useful to Evaluate the Down-Regulation of SCD GeneExpression

Cell Culture

There are numerous cell culture systems that can be used to analyzereduction of SCD levels either directly or indirectly by measuringdownstream effects. For example, cultured HepG2, MDA-MB-231 or A549cells can be used in cell culture experiments to assess the efficacy ofnucleic acid molecules of the invention. As such, cells treated withnucleic acid molecules of the invention (e.g., siNA) targeting SCD RNAwould be expected to have decreased SCD expression capacity compared tomatched control nucleic acid molecules having a scrambled or inactivesequence. In a non-limiting example, cells are cultured and SCDexpression is quantified, for example, by time-resolved immunofluorometric assay. SCD messenger-RNA expression is quantitated withRT-PCR in cultured cells. Untreated cells are compared to cells treatedwith siNA molecules transfected with a suitable reagent, for example acationic lipid such as lipofectamine, and SCD protein and RNA levels arequantitated. Dose response assays are then performed to establish dosedependent inhibition of SCD expression.

Liver stearoyl-CoA desaturase (SCD) activity has been implicated inexcessive adiposity in chickens. Studies suggest that the difference inSCD activity between fat and lean chickens is explained by a differencein SCD I gene expression. (Lefevre et al., 1999, Archives ofBiochemistry and Biophysics, 368, 329-337). Lefevre et al. describe astudy using primary cultures of 6-week-old chicken hepatocytes, in whichSCD1 gene expression was analyzed as the result of insulin and glucagonaction. The studies showed that insulin increased SCD1 activity and mRNAlevels, whereas glucagon dramatically decreased both SCD1 enzymeactivity and SCD1 mRNA levels. Nuclear run-on transcription assays andmRNA stability investigations demonstrated that insulin and glucagoneffects on SCD1 gene expression were primarily transcriptional innature. Furthermore, the results indicated that the glucagon-mediatedinhibition of SCD1 gene transcription was more potent than simplycounteracting the insulin-mediated effect. Moreover, among hepatic genesinvolved in lipid metabolism in chickens, SCD1 was the first gene shownto be regulated at the transcriptional level by insulin, in the absenceof triiodothyronine. These studies demonstrate that the growing chickenhepatocyte culture model is a useful model in studying the effect of SCDinhibitors, including siNA molecules of the present invention.

In several cell culture systems, cationic lipids have been shown toenhance the bioavailability of oligonucleotides to cells in culture(Bennet, et al., 1992, Mol. Pharmacology, 41, 1023-1033). In oneembodiment, siNA molecules of the invention are complexed with cationiclipids for cell culture experiments. siNA and cationic lipid mixturesare prepared in serum-free DMEM immediately prior to addition to thecells. DMEM plus additives are warmed to room temperature (about 20-25°C.) and cationic lipid is added to the final desired concentration andthe solution is vortexed briefly. siNA molecules are added to the finaldesired concentration and the solution is again vortexed briefly andincubated for 10 minutes at room temperature. In dose responseexperiments, the RNA/lipid complex is serially diluted into DMEMfollowing the 10 minute incubation.

Evaluating the efficacy of anti-SCD agents in animal models is animportant prerequisite to human clinical trials. Stearoyl-CoA desaturase(SCD) mRNA is overexpressed in various animal models of obesity and type2 diabetes, see for example Ntambi et al., 2000, Biomedical and HealthResearch, 37, 69-78 and Miyazaki et al., 2001, J. Biol. Chem., 276,39455-39461. SCD expression is known to dramatically increase during thedifferentiation of preadipocytes into adipocytes. Thiazolidinediones(TZDs), ligands for the adipocyte-specific nuclear peroxisomeproliferator-activated receptor gamma 2 (PPAR-gamma-2), enhance theconversion of preadipocytes into mature adipocytes in vivo and in vitro,and can exert potent antidiabetic effects by enhancing sensitivity toinsulin in target tissues. TZDs have been shown to repress theexpression of the SCD1 gene isoform in differentiating preadipocytes andmature adipocytes (Ntambi et al., 2000, Biomedical and Health Research,37, 69-78). The repression of several other adipogenic genes, includingthe SCD2 isoform, were not affected by TZDs. In differentiatingpreadipocytes and mature adipocytes, the downregulation of the SCD1 geneexpression was accompanied by a decrease in SCD protein and enzymeactivity, as well as a dramatic decrease in palmitoleate (C16:1n-7)composition. The adipocytes were smaller in size and contained smallerfat droplets as observed by Oil Red O staining. These data indicate thatthe TZDs specifically target SCD1 gene expression in bothdifferentiating and mature adipocytes, which consequently decreases theC16:1n-7 composition of triglycerides. The models described by Ntambi etal., 2000, Biomedical and Health Research, 37, 69-78; Miyazaki et al.,2001, J. Biol. Chem., 276, 39455-39461; and Zheng et al., 1999, NatureGenetics, 23, 268-270, such as asebia mouse strains (ab^(j) and ab^(2j))and mouse models with a targeted disruption (SCD1 −/−) can be used tostudy the effect of SCD inhibition using siNA molecules of theinvention.

Example 9 RNAi Mediated Inhibition of SCD Expression

siNA constructs (Table III) are tested for efficacy in reducing SCD RNAexpression in, for example, HepG2, MDA-MB-231 or A549 cells. Cells areplated approximately 24 hours before transfection in 96-well plates at5,000-7,500 cells/well, 100 μl/well, such that at the time oftransfection cells are 70-90% confluent. For transfection, annealedsiNAs are mixed with the transfection reagent (Lipofectamine 2000,Invitrogen) in a volume of 50 μl/well and incubated for 20 minutes atroom temperature. The siNA transfection mixtures are added to cells togive a final siNA concentration of 25 nM in a volume of 150 μl. EachsiNA transfection mixture is added to 3 wells for triplicate siNAtreatments. Cells are incubated at 37° for 24 hours in the continuedpresence of the siNA transfection mixture. At 24 hours, RNA is preparedfrom each well of treated cells. The supernatants with the transfectionmixtures are first removed and discarded, then the cells are lysed andRNA prepared from each well. Target gene expression following treatmentis evaluated by RT-PCR for the target gene and for a control gene (36B4,an RNA polymerase subunit) for normalization. The triplicate data isaveraged and the standard deviations determined for each treatment.Normalized data are graphed and the percent reduction of target mRNA byactive siNAs in comparison to their respective inverted control siNAs isdetermined.

In a non-limiting example, chemically modified siNA constructs (TableIII) were tested for efficacy as described above in reducing SCD RNAexpression in A549 cells. Active siNAs were evaluated compared tountreated cells, a matched chemistry irrelevant control (IC,32072/32075), and a transfection control. Results are summarized in FIG.22. FIG. 22 shows results for chemically modified siNA constructstargeting various sites in SCD mRNA. As shown in FIG. 22, the activesiNA constructs provide significant inhibition of SCD gene expression incell culture experiments as determined by levels of SCD mRNA whencompared to appropriate controls.

Example 10 Indications

The present body of knowledge in SCD research indicates the need formethods to assay SCD activity and for compounds that can regulate SCDexpression for research, diagnostic, and therapeutic use. As describedherein, the nucleic acid molecules of the present invention can be usedin assays to diagnose disease state related of SCD levels. In addition,the nucleic acid molecules can be used to treat disease state related toSCD levels.

Particular conditions and disease states that can be associated with SCDexpression modulation include, but are not limited to diabetes (type Iand/or type II), atherosclerosis, cancer, obesity, and viral infection,and any other diseases, conditions or disorders that are related to orwill respond to the levels of SCD in a cell or tissue, alone or incombination with other therapies.

Thiazolidinediones (TZDs), insulin, and PTP-1B inhibitors (see forexample McSwiggen, U.S. Ser. No. 10/206,705) are non-limiting examplesof pharmaceutical agents that can be combined with or used inconjunction with the nucleic acid molecules (e.g. siRNA molecules) ofthe instant invention. Those skilled in the art will recognize thatother drugs, such as anti-diabetes and anti-obesity compounds andtherapies, can similarly be readily combined with the nucleic acidmolecules of the instant invention (e.g. siRNA molecules) and are hencewithin the scope of the instant invention.

Example 11 Diagnostic Uses

The siNA molecules of the invention can be used in a variety ofdiagnostic applications, such as in the identification of moleculartargets (e.g., RNA) in a variety of applications, for example, inclinical, industrial, environmental, agricultural and/or researchsettings. Such diagnostic use of siNA molecules involves utilizingreconstituted RNAi systems, for example, using cellular lysates orpartially purified cellular lysates. siNA molecules of this inventioncan be used as diagnostic tools to examine genetic drift and mutationswithin diseased cells or to detect the presence of endogenous orexogenous, for example viral, RNA in a cell. The close relationshipbetween siNA activity and the structure of the target RNA allows thedetection of mutations in any region of the molecule, which alters thebase-pairing and three-dimensional structure of the target RNA. By usingmultiple siNA molecules described in this invention, one can mapnucleotide changes, which are important to RNA structure and function invitro, as well as in cells and tissues. Cleavage of target RNAs withsiNA molecules can be used to inhibit gene expression and define therole of specified gene products in the progression of disease orinfection. In this manner, other genetic targets can be defined asimportant mediators of the disease. These experiments will lead tobetter treatment of the disease progression by affording the possibilityof combination therapies (e.g., multiple siNA molecules targeted todifferent genes, siNA molecules coupled with known small moleculeinhibitors, or intermittent treatment with combinations siNA moleculesand/or other chemical or biological molecules). Other in vitro uses ofsiNA molecules of this invention are well known in the art, and includedetection of the presence of mRNAs associated with a disease, infection,or related condition. Such RNA is detected by determining the presenceof a cleavage product after treatment with a siNA using standardmethodologies, for example, fluorescence resonance emission transfer(FRET).

In a specific example, siNA molecules that cleave only wild-type ormutant forms of the target RNA are used for the assay. The first siNAmolecules (i.e., those that cleave only wild-type forms of target RNA)are used to identify wild-type RNA present in the sample and the secondsiNA molecules (i.e., those that cleave only mutant forms of target RNA)are used to identify mutant RNA in the sample. As reaction controls,synthetic substrates of both wild-type and mutant RNA are cleaved byboth siNA molecules to demonstrate the relative siNA efficiencies in thereactions and the absence of cleavage of the “non-targeted” RNA species.The cleavage products from the synthetic substrates also serve togenerate size markers for the analysis of wild-type and mutant RNAs inthe sample population. Thus, each analysis requires two siNA molecules,two substrates and one unknown sample, which is combined into sixreactions. The presence of cleavage products is determined using anRNase protection assay so that full-length and cleavage fragments ofeach RNA can be analyzed in one lane of a polyacrylamide gel. It is notabsolutely required to quantify the results to gain insight into theexpression of mutant RNAs and putative risk of the desired phenotypicchanges in target cells. The expression of mRNA whose protein product isimplicated in the development of the phenotype (i.e., disease related orinfection related) is adequate to establish risk. If probes ofcomparable specific activity are used for both transcripts, then aqualitative comparison of RNA levels is adequate and decreases the costof the initial diagnosis. Higher mutant form to wild-type ratios arecorrelated with higher risk whether RNA levels are comparedqualitatively or quantitatively.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims. The present invention teaches oneskilled in the art to test various combinations and/or substitutions ofchemical modifications described herein toward generating nucleic acidconstructs with improved activity for mediating RNAi activity. Suchimproved activity can comprise improved stability, improvedbioavailability, and/or improved activation of cellular responsesmediating RNAi. Therefore, the specific embodiments described herein arenot limiting and one skilled in the art can readily appreciate thatspecific combinations of the modifications described herein can betested without undue experimentation toward identifying siNA moleculeswith improved RNAi activity.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments, optional features, modification and variation ofthe concepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group. TABLE I SCD Accession Numbers XM_208174 Homosapiens similar to stearoyl-CoA desaturase (delta-9-desaturase) [Homosapiens] (LOC284202), mRNA gi|27500564|ref|XM_208174.1|[27500564]NM_005063 Homo sapiens stearoyl-CoA desaturase (delta-9- desaturase)(SCD), mRNA gi|19923295|ref|NM_005063.2|[19923295] Y13647 Homo sapiensmRNA for stearoyl-CoA desaturasegi|2190403|emb|Y13647.1|HSSTEACOA[2190403] AF320307 Homo sapiensstearoyl-CoA desaturase (SCD) gene, exon 1 and intron 1 and partial cdsgi|14150490|gb|AF320307.1|AF320307[14150490] AU098830 AU098830 SuganoHomo sapiens cDNA library Homo sapiens cDNA clone HEP02242 similar toHomo sapiens stearoyl-CoA desaturase (SCD) mRNA, MRNA sequencegi|13549959|dbj|AU098830.1|[13549959] BG319495 Tmdh05 Human KeratinocyteSubtraction Library- Downregulated Transcripts Homo sapiens cDNA similarto stearoyl-CoA desaturase (delta-9-desaturase) (SCD), MRNA sequencegi|13129361|gb|BG319495.1|[13129361] BG319433 4dh85 Human KeratinocyteSubtraction Library- Downregulated Transcripts Homo sapiens cDNA similarto stearoyl-CoA desaturase (SCD), MRNA sequencegi|13129299|gb|BG319433.1|[13129299] AU076658 AU076658 Sugano cDNAlibrary Homo sapiens cDNA clone HEP02242 similar to 5′-end region ofHomo sapiens mRNA for stearoyl-CoA desaturase, MRNA sequencegi|7439136|dbj|AU076658.1|[7439136] AB032261 Homo sapiens Scd mRNA forstearoyl-CoA desaturase, complete cdsgi|7415720|dbj|AB032261.1|[7415720] AF097514 Homo sapiens stearoyl-CoAdesaturase (SCD) mRNA, complete cdsgi|4808600|gb|AF097514.1|AF097514[4808600] S70284 stearoyl-CoAdesaturase [human, adipose tissue, mRNA Partial, 712 nt]gi|546517|bbm|339737|bbs|148167|gb|S70284.1|S70284[546517]

TABLE II SCD siNA AND TARGET SEQUENCES SCD NM_005063.2 Pos Seq Seq IDUPos Upper seq Seq ID LPos Lower seq Seq ID 3 AAAAGGGGGCUGAGGAAAU 1 3AAAAGGGGGCUGAGGAAAU 1 21 AUUUCCUCAGCCCCCUUUU 291 21 UACCGGACACGGUCACCCG2 21 UACCGGACACGGUCACCCG 2 39 CGGGUGACCGUGUCCGGUA 292 39GUUGCCAGCUCUAGCCUUU 3 39 GUUGCCAGCUCUAGCCUUU 3 57 AAAGGCUAGAGCUGGCAAC293 57 UAAAUUCCCGGCUCGGGGA 4 57 UAAAUUCCCGGCUCGGGGA 4 75UCCCCGAGCCGGGAAUUUA 294 75 ACCUCCACGCACCGCGGCU 5 75 ACCUCCACGCACCGCGGCU5 93 AGCCGCGGUGCGUGGAGGU 295 93 UAGCGCCGACAACCAGCUA 6 93UAGCGCCGACAACCAGCUA 6 111 UAGCUGGUUGUCGGCGCUA 296 111AGCGUGCAAGGCGCCGCGG 7 111 AGCGUGCAAGGCGCCGCGG 7 129 CCGCGGCGCCUUGCACGCU297 129 GCUCAGCGCGUACCGGCGG 8 129 GCUCAGCGCGUACCGGCGG 8 147CCGCCGGUACGCGCUGAGC 298 147 GGCUUCGAAACCGCAGUCC 9 147GGCUUCGAAACCGCAGUCC 9 165 GGACUGCGGUUUCGAAGCC 299 165CUCCGGCGACCCCGAACUC 10 165 CUCCGGCGACCCCGAACUC 10 183GAGUUCGGGGUCGCCGGAG 300 183 CCGCUCCGGAGCCUCAGCC 11 183CCGCUCCGGAGCCUCAGCC 11 201 GGCUGAGGCUCCGGAGCGG 301 201CCCCUGGAAAGUGAUCCCG 12 201 CCCCUGGAAAGUGAUCCCG 12 219CGGGAUCACUUUCCAGGGG 302 219 GGCAUCCGAGAGCCAAGAU 13 219GGCAUCCGAGAGCCAAGAU 13 237 AUCUUGGCUCUCGGAUGCC 303 237UGCCGGCCCACUUGCUGCA 14 237 UGCCGGCCCACUUGCUGCA 14 255UGCAGCAAGUGGGCCGGCA 304 255 AGGACGAUAUCUCUAGCUC 15 255AGGACGAUAUCUCUAGCUC 15 273 GAGCUAGAGAUAUCGUCCU 305 273CCUAUACCACCACCACCAC 16 273 CCUAUACCACCACCACCAC 16 291GUGGUGGUGGUGGUAUAGG 306 291 CCAUUACAGCGCCUCCCUC 17 291CCAUUACAGCGCCUCCCUC 17 309 GAGGGAGGCGCUGUAAUGG 307 309CCAGGGUCCUGCAGAAUGG 18 309 CCAGGGUCCUGCAGAAUGG 18 327CCAUUCUGCAGGACCCUGG 308 327 GAGGAGAUAAGUUGGAGAC 19 327GAGGAGAUAAGUUGGAGAC 19 345 GUCUCCAACUUAUCUCCUC 309 345CGAUGCCCCUCUACUUGGA 20 345 CGAUGCCCCUCUACUUGGA 20 363UCCAAGUAGAGGGGCAUCG 310 363 AAGACGACAUUCGCCCUGA 21 363AAGACGACAUUCGCCCUGA 21 381 UCAGGGCGAAUGUCGUCUU 311 381AUAUAAAAGAUGAUAUAUA 22 381 AUAUAAAAGAUGAUAUAUA 22 399UAUAUAUCAUCUUUUAUAU 312 399 AUGACCCCACCUACAAGGA 23 399AUGACCCCACCUACAAGGA 23 417 UCCUUGUAGGUGGGGUCAU 313 417AUAAGGAAGGCCCAAGCCC 24 417 AUAAGGAAGGCCCAAGCCC 24 435GGGCUUGGGCCUUCCUUAU 314 435 CCAAGGUUGAAUAUGUCUG 25 435CCAAGGUUGAAUAUGUCUG 25 453 CAGACAUAUUCAACCUUGG 315 453GGAGAAACAUCAUCCUUAU 26 453 GGAGAAACAUCAUCCUUAU 26 471AUAAGGAUGAUGUUUCUCC 316 471 UGUCUCUGCUACACUUGGG 27 471UGUCUCUGCUACACUUGGG 27 489 CCCAAGUGUAGCAGAGACA 317 489GAGCCCUGUAUGGGAUCAC 28 489 GAGCCCUGUAUGGGAUCAC 28 507GUGAUCCCAUACAGGGCUC 318 507 CUUUGAUUCCUACCUGCAA 29 507CUUUGAUUCCUACCUGCAA 29 525 UUGCAGGUAGGAAUCAAAG 319 525AGUUCUACACCUGGCUUUG 30 525 AGUUCUACACCUGGCUUUG 30 543CAAAGCCAGGUGUAGAACU 320 543 GGGGGGUAUUCUACUAUUU 31 543GGGGGGUAUUCUACUAUUU 31 561 AAAUAGUAGAAUACCCCCC 321 561UUGUCAGUGCCCUGGGCAU 32 561 UUGUCAGUGCCCUGGGCAU 32 579AUGCCCAGGGCACUGACAA 322 579 UAACAGCAGGAGCUCAUCG 33 579UAACAGCAGGAGCUCAUCG 33 597 CGAUGAGCUCCUGCUGUUA 323 597GUCUGUGGAGCCACCGCUC 34 597 GUCUGUGGAGCCACCGCUC 34 615GAGCGGUGGCUCCACAGAC 324 615 CUUACAAAGCUCGGCUGCC 35 615CUUACAAAGCUCGGCUGCC 35 633 GGCAGCCGAGCUUUGUAAG 325 633CCCUACGGCUCUUUCUGAU 36 633 CCCUACGGCUCUUUCUGAU 36 651AUCAGAAAGAGCCGUAGGG 326 651 UCAUUGCCAACACAAUGGC 37 651UCAUUGCCAACACAAUGGC 37 669 GCCAUUGUGUUGGCAAUGA 327 669CAUUCCAGAAUGAUGUCUA 38 669 CAUUCCAGAAUGAUGUCUA 38 687UAGACAUCAUUCUGGAAUG 328 687 AUGAAUGGGCUCGUGACCA 39 687AUGAAUGGGCUCGUGACCA 39 705 UGGUCACGAGCCCAUUCAU 329 705ACCGUGCCCACCACAAGUU 40 705 ACCGUGCCCACCACAAGUU 40 723AACUUGUGGUGGGCACGGU 330 723 UUUCAGAAACACAUGCUGA 41 723UUUCAGAAACACAUGCUGA 41 741 UCAGCAUGUGUUUCUGAAA 331 741AUCCUCAUAAUUCCCGACG 42 741 AUCCUCAUAAUUCCCGACG 42 759CGUCGGGAAUUAUGAGGAU 332 759 GUGGCUUUUUCUUCUCUCA 43 759GUGGCUUUUUCUUCUCUCA 43 777 UGAGAGAAGAAAAAGCCAC 333 777ACGUGGGUUGGCUGCUUGU 44 777 ACGUGGGUUGGCUGCUUGU 44 795ACAAGCAGCCAACCCACGU 334 795 UGCGCAAACACCCAGCUGU 45 795UGCGCAAACACCCAGCUGU 45 813 ACAGCUGGGUGUUUGCGCA 335 813UCAAAGAGAAGGGGAGUAC 46 813 UCAAAGAGAAGGGGAGUAC 46 831GUACUCCCCUUCUCUUUGA 336 831 CGCUAGACUUGUCUGACCU 47 831CGCUAGACUUGUCUGACCU 47 849 AGGUCAGACAAGUCUAGCG 337 849UAGAAGCUGAGAAACUGGU 48 849 UAGAAGCUGAGAAACUGGU 48 867ACCAGUUUCUCAGCUUCUA 338 867 UGAUGUUCCAGAGGAGGUA 49 867UGAUGUUCCAGAGGAGGUA 49 885 UACCUCCUCUGGAACAUCA 339 885ACUACAAACCUGGCUUGCU 50 885 ACUACAAACCUGGCUUGCU 50 903AGCAAGCCAGGUUUGUAGU 340 903 UGCUGAUGUGCUUCAUCCU 51 903UGCUGAUGUGCUUCAUCCU 51 921 AGGAUGAAGCACAUCAGCA 341 921UGCCCACGCUUGUGCCCUG 52 921 UGCCCACGCUUGUGCCCUG 52 939CAGGGCACAAGCGUGGGCA 342 939 GGUAUUUCUGGGGUGAAAC 53 939GGUAUUUCUGGGGUGAAAC 53 957 GUUUCACCCCAGAAAUACC 343 957CUUUUCAAAACAGUGUGUU 54 957 CUUUUCAAAACAGUGUGUU 54 975AACACACUGUUUUGAAAAG 344 975 UCGUUGCCACUUUCUUGCG 55 975UCGUUGCCACUUUCUUGCG 55 993 CGCAAGAAAGUGGCAACGA 345 993GAUAUGCUGUGGUGCUUAA 56 993 GAUAUGCUGUGGUGCUUAA 56 1011UUAAGCACCACAGCAUAUC 346 1011 AUGCCACCUGGCUGGUGAA 57 1011AUGCCACCUGGCUGGUGAA 57 1029 UUCACCAGCCAGGUGGCAU 347 1029ACAGUGCUGCCCACCUCUU 58 1029 ACAGUGCUGCCCACCUCUU 58 1047AAGAGGUGGGCAGCACUGU 348 1047 UCGGAUAUCGUCCUUAUGA 59 1047UCGGAUAUCGUCCUUAUGA 59 1065 UCAUAAGGACGAUAUCCGA 349 1065ACAAGAACAUUAGCCCCCG 60 1065 ACAAGAACAUUAGCCCCCG 60 1083CGGGGGCUAAUGUUCUUGU 350 1083 GGGAGAAUAUCCUGGUUUC 61 1083GGGAGAAUAUCCUGGUUUC 61 1101 GAAACCAGGAUAUUCUCCC 351 1101CACUUGGAGCUGUGGGUGA 62 1101 CACUUGGAGCUGUGGGUGA 62 1119UCACCCACAGCUCCAAGUG 352 1119 AGGGCUUCCACAACUACCA 63 1119AGGGCUUCCACAACUACCA 63 1137 UGGUAGUUGUGGAAGCCCU 353 1137ACCACUCCUUUCCCUAUGA 64 1137 ACCACUCCUUUCCCUAUGA 64 1155UCAUAGGGAAAGGAGUGGU 354 1155 ACUACUCUGCCAGUGAGUA 65 1155ACUACUCUGCCAGUGAGUA 65 1173 UACUCACUGGCAGAGUAGU 355 1173ACCGCUGGCACAUCAACUU 66 1173 ACCGCUGGCACAUCAACUU 66 1191AAGUUGAUGUGCCAGCGGU 356 1191 UCACCACAUUCUUCAUUGA 67 1191UCACCACAUUCUUCAUUGA 67 1209 UCAAUGAAGAAUGUGGUGA 357 1209AUUGCAUGGCCGCCCUCGG 68 1209 AUUGCAUGGCCGCCCUCGG 68 1227CCGAGGGCGGCCAUGCAAU 358 1227 GUCUGGCCUAUGACCGGAA 69 1227GUCUGGCCUAUGACCGGAA 69 1245 UUCCGGUCAUAGGCCAGAC 359 1245AGAAAGUCUCCAAGGCCGC 70 1245 AGAAAGUCUCCAAGGCCGC 70 1263GCGGCCUUGGAGACUUUCU 360 1263 CCAUCUUGGCCAGGAUUAA 71 1263CCAUCUUGGCCAGGAUUAA 71 1281 UUAAUCCUGGCCAAGAUGG 361 1281AAAGAACCGGAGAUGGAAA 72 1281 AAAGAACCGGAGAUGGAAA 72 1299UUUCCAUCUCCGGUUCUUU 362 1299 ACUACAAGAGUGGCUGAGU 73 1299ACUACAAGAGUGGCUGAGU 73 1317 ACUCAGCCACUCUUGUAGU 363 1317UUUGGGGUCCCUCAGGUUU 74 1317 UUUGGGGUCCCUCAGGUUU 74 1335AAACCUGAGGGACCCCAAA 364 1335 UCCUUUUUCAAAAACCAGC 75 1335UCCUUUUUCAAAAACCAGC 75 1353 GCUGGUUUUUGAAAAAGGA 365 1353CCAGGCAGAGGUUUUAAUG 76 1353 CCAGGCAGAGGUUUUAAUG 76 1371CAUUAAAACCUCUGCCUGG 366 1371 GUCUGUUUAUUAACUACUG 77 1371GUCUGUUUAUUAACUACUG 77 1389 CAGUAGUUAAUAAACAGAC 367 1389GAAUAAUGCUACCAGGAUG 78 1389 GAAUAAUGCUACCAGGAUG 78 1407CAUCCUGGUAGCAUUAUUC 368 1407 GCUAAAGAUGAUGAUGUUA 79 1407GCUAAAGAUGAUGAUGUUA 79 1425 UAACAUCAUCAUCUUUAGC 369 1425AACCCAUUCCAGUACAGUA 80 1425 AACCCAUUCCAGUACAGUA 80 1443UACUGUACUGGAAUGGGUU 370 1443 AUUCUUUUAAAAUUCAAAA 81 1443AUUCUUUUAAAAUUCAAAA 81 1461 UUUUGAAUUUUAAAAGAAU 371 1461AGUAUUGAAAGCCAACAAC 82 1461 AGUAUUGAAAGCCAACAAC 82 1479GUUGUUGGCUUUCAAUACU 372 1479 CUCUGCCUUUAUGAUGCUA 83 1479CUCUGCCUUUAUGAUGCUA 83 1497 UAGCAUCAUAAAGGCAGAG 373 1497AAGCUGAUAUUAUUUCUUC 84 1497 AAGCUGAUAUUAUUUCUUC 84 1515GAAGAAAUAAUAUCAGCUU 374 1515 CUCUUAUCCUCUCUCUCUU 85 1515CUCUUAUCCUCUCUCUCUU 85 1533 AAGAGAGAGAGGAUAAGAG 375 1533UCUAGGCCCAUUGUCCUCC 86 1533 UCUAGGCCCAUUGUCCUCC 86 1551GGAGGACAAUGGGCCUAGA 376 1551 CUUUUCACUUUAUUGCUAU 87 1551CUUUUCACUUUAUUGCUAU 87 1569 AUAGCAAUAAAGUGAAAAG 377 1569UCGCCCUCCUUUCCCUUAU 88 1569 UCGCCCUCCUUUCCCUUAU 88 1587AUAAGGGAAAGGAGGGCGA 378 1587 UUGCCUCCCAGGCAAGCAG 89 1587UUGCCUCCCAGGCAAGCAG 89 1605 CUGCUUGCCUGGGAGGCAA 379 1605GCUGGUCAGUCUUUGCUCA 90 1605 GCUGGUCAGUCUUUGCUCA 90 1623UGAGCAAAGACUGACCAGC 380 1623 AGUGUCCAGCUUCCAAAGC 91 1623AGUGUCCAGCUUCCAAAGC 91 1641 GCUUUGGAAGCUGGACACU 381 1641CCUAGACAACCUUUCUGUA 92 1641 CCUAGACAACCUUUCUGUA 92 1659UACAGAAAGGUUGUCUAGG 382 1659 AGCCUAAAACGAAUGGUCU 93 1659AGCCUAAAACGAAUGGUCU 93 1677 AGACCAUUCGUUUUAGGCU 383 1677UUUGCUCCAGAUAACUCUC 94 1677 UUUGCUCCAGAUAACUCUC 94 1695GAGAGUUAUCUGGAGCAAA 384 1695 CUUUCCUUGAGCUGUUGUG 95 1695CUUUCCUUGAGCUGUUGUG 95 1713 CACAACAGCUCAAGGAAAG 385 1713GAGCUUUGAAGUAGGUGGC 96 1713 GAGCUUUGAAGUAGGUGGC 96 1731GCCACCUACUUCAAAGCUC 386 1731 CUUGAGCUAGAGAUAAAAC 97 1731CUUGAGCUAGAGAUAAAAC 97 1749 GUUUUAUCUCUAGCUCAAG 387 1749CAGAAUCUUCUGGGUAGUC 98 1749 CAGAAUCUUCUGGGUAGUC 98 1767GACUACCCAGAAGAUUCUG 388 1767 CCCCUGUUGAUUAUCUUCA 99 1767CCCCUGUUGAUUAUCUUCA 99 1785 UGAAGAUAAUCAACAGGGG 389 1785AGCCCAGGCUUUUGCUAGA 100 1785 AGCCCAGGCUUUUGCUAGA 100 1803UCUAGCAAAAGCCUGGGCU 390 1803 AUGGAAUGGAAAAGCAACU 101 1803AUGGAAUGGAAAAGCAACU 101 1821 AGUUGCUUUUCCAUUCCAU 391 1821UUCAUUUGACACAAAGCUU 102 1821 UUCAUUUGACACAAAGCUU 102 1839AAGCUUUGUGUCAAAUGAA 392 1839 UCUAAAGCAGGUAAAUUGU 103 1839UCUAAAGCAGGUAAAUUGU 103 1857 ACAAUUUACCUGCUUUAGA 393 1857UCGGGGGAGAGAGUUAGCA 104 1857 UCGGGGGAGAGAGUUAGCA 104 1875UGCUAACUCUCUCCCCCGA 394 1875 AUGUAUGAAUGUAAGGAUG 105 1875AUGUAUGAAUGUAAGGAUG 105 1893 CAUCCUUACAUUCAUACAU 395 1893GAGGGAAGCGAAGCAAGAG 106 1893 GAGGGAAGCGAAGCAAGAG 106 1911CUCUUGCUUCGCUUCCCUC 396 1911 GGAACCUCUCGCCAUGAUC 107 1911GGAACCUCUCGCCAUGAUC 107 1929 GAUCAUGGCGAGAGGUUCC 397 1929CAGACAUACAGCUGCCUAC 108 1929 CAGACAUACAGCUGCCUAC 108 1947GUAGGCAGCUGUAUGUCUG 398 1947 CCUAAUGAGGACUUCAAGC 109 1947CCUAAUGAGGACUUCAAGC 109 1965 GCUUGAAGUCCUCAUUAGG 399 1965CCCCACCACAUAGCAUGCU 110 1965 CCCCACCACAUAGCAUGCU 110 1983AGCAUGCUAUGUGGUGGGG 400 1983 UUCCUUUCUCUCCUGGCUC 111 1983UUCCUUUCUCUCCUGGCUC 111 2001 GAGCCAGGAGAGAAAGGAA 401 2001CGGGGUAAAAAGUGGCUGC 112 2001 CGGGGUAAAAAGUGGCUGC 112 2019GCAGCCACUUUUUACCCCG 402 2019 CGGUGUUUGGCAAUGCUAA 113 2019CGGUGUUUGGCAAUGCUAA 113 2037 UUAGCAUUGCCAAACACCG 403 2037AUUCAAUGCCGCAACAUAU 114 2037 AUUCAAUGCCGCAACAUAU 114 2055AUAUGUUGCGGCAUUGAAU 404 2055 UAGUUGAGGCCGAGGAUAA 115 2055UAGUUGAGGCCGAGGAUAA 115 2073 UUAUCCUCGGCCUCAACUA 405 2073AAGAAAAGACAUUUUAAGU 116 2073 AAGAAAAGACAUUUUAAGU 116 2091ACUUAAAAUGUCUUUUCUU 406 2091 UUUGUAGUAAAAGUGGUCU 117 2091UUUGUAGUAAAAGUGGUCU 117 2109 AGACCACUUUUACUACAAA 407 2109UCUGCUGGGGAAGGGUUUU 118 2109 UCUGCUGGGGAAGGGUUUU 118 2127AAAACCCUUCCCCAGCAGA 408 2127 UCUUUUCUUUUUUUCUUUA 119 2127UCUUUUCUUUUUUUCUUUA 119 2145 UAAAGAAAAAAAGAAAAGA 409 2145AAUAACAAGGAGAUUUCUU 120 2145 AAUAACAAGGAGAUUUCUU 120 2163AAGAAAUCUCCUUGUUAUU 410 2163 UAGUUCAUAUAUCAAGAAG 121 2163UAGUUCAUAUAUCAAGAAG 121 2181 CUUCUUGAUAUAUGAACUA 411 2181GUCUUGAAGUUGGGUGUUU 122 2181 GUCUUGAAGUUGGGUGUUU 122 2199AAACACCCAACUUCAAGAC 412 2199 UCCAGAAUUGGUAAAAACA 123 2199UCCAGAAUUGGUAAAAACA 123 2217 UGUUUUUACCAAUUCUGGA 413 2217AGCAGCUCAUGGAAUUUUG 124 2217 AGCAGCUCAUGGAAUUUUG 124 2235CAAAAUUCCAUGAGCUGCU 414 2235 GAGUAUUCCAUGAGCUGCU 125 2235GAGUAUUCCAUGAGCUGCU 125 2253 AGCAGCUCAUGGAAUACUC 415 2253UCAUUACAGUUCUUUCCUC 126 2253 UCAUUACAGUUCUUUCCUC 126 2271GAGGAAAGAACUGUAAUGA 416 2271 CUUUCUGCUCUGCCAUCUU 127 2271CUUUCUGCUCUGCCAUCUU 127 2289 AAGAUGGCAGAGCAGAAAG 417 2289UCAGGAUAUUGGUUCUUCC 128 2289 UCAGGAUAUUGGUUCUUCC 128 2307GGAAGAACCAAUAUCCUGA 418 2307 CCCUCAUAGUAAUAAGAUG 129 2307CCCUCAUAGUAAUAAGAUG 129 2325 CAUCUUAUUACUAUGAGGG 419 2325GGCUGUGGCAUUUCCAAAC 130 2325 GGCUGUGGCAUUUCCAAAC 130 2343GUUUGGAAAUGCCACAGCC 420 2343 CAUCCAAAAAAAGGGAAGG 131 2343CAUCCAAAAAAAGGGAAGG 131 2361 CCUUCCCUUUUUUUGGAUG 421 2361GAUUUAAGGAGGUGAAGUC 132 2361 GAUUUAAGGAGGUGAAGUC 132 2379GACUUCACCUCCUUAAAUC 422 2379 CGGGUCAAAAAUAAAAUAU 133 2379CGGGUCAAAAAUAAAAUAU 133 2397 AUAUUUUAUUUUUGACCCG 423 2397UAUAUACAUAUAUACAUUG 134 2397 UAUAUACAUAUAUACAUUG 134 2415CAAUGUAUAUAUGUAUAUA 424 2415 GCUUAGAACGUUAAACUAU 135 2415GCUUAGAACGUUAAACUAU 135 2433 AUAGUUUAACGUUCUAAGC 425 2433UUAGAGUAUUUCCCUUCCA 136 2433 UUAGAGUAUUUCCCUUCCA 136 2451UGGAAGGGAAAUACUCUAA 426 2451 AAAGAGGGAUGUUUGGAAA 137 2451AAAGAGGGAUGUUUGGAAA 137 2469 UUUCCAAACAUCCCUCUUU 427 2469AAAACUCUGAAGGAGAGGA 138 2469 AAAACUCUGAAGGAGAGGA 138 2487UCCUCUCCUUCAGAGUUUU 428 2487 AGGAAUUAGUUGGGAUGCC 139 2487AGGAAUUAGUUGGGAUGCC 139 2505 GGCAUCCCAACUAAUUCCU 429 2505CAAUUUCCUCUCCACUGCU 140 2505 CAAUUUCCUCUCCACUGCU 140 2523AGCAGUGGAGAGGAAAUUG 430 2523 UGGACAUGAGAUGGAGAGG 141 2523UGGACAUGAGAUGGAGAGG 141 2541 CCUCUCCAUCUCAUGUCCA 431 2541GCUGAGGGACAGGAUCUAU 142 2541 GCUGAGGGACAGGAUCUAU 142 2559AUAGAUCCUGUCCCUCAGC 432 2559 UAGGCAGCUUCUAAGAGCG 143 2559UAGGCAGCUUCUAAGAGCG 143 2577 CGCUCUUAGAAGCUGCCUA 433 2577GAACUUCACAUAGGAAGGG 144 2577 GAACUUCACAUAGGAAGGG 144 2595CCCUUCCUAUGUGAAGUUC 434 2595 GAUCUGAGAACACGUUGCC 145 2595GAUCUGAGAACACGUUGCC 145 2613 GGCAACGUGUUCUCAGAUC 435 2613CAGGGGCUUGAGAAGGUUA 146 2613 CAGGGGCUUGAGAAGGUUA 146 2631UAACCUUCUCAAGCCCCUG 436 2631 ACUGAGUGAGUUAUUGGGA 147 2631ACUGAGUGAGUUAUUGGGA 147 2649 UCCCAAUAACUCACUCAGU 437 2649AGUCUUAAUAAAAUAAACU 148 2649 AGUCUUAAUAAAAUAAACU 148 2667AGUUUAUUUUAUUAAGACU 438 2667 UAGAUAUUAGGUCCAUUCA 149 2667UAGAUAUUAGGUCCAUUCA 149 2685 UGAAUGGACCUAAUAUCUA 439 2685AUUAAUUAGUUCCAGUUUC 150 2685 AUUAAUUAGUUCCAGUUUC 150 2703GAAACUGGAACUAAUUAAU 440 2703 CUCCUUGAAAUGAGUAAAA 151 2703CUCCUUGAAAUGAGUAAAA 151 2721 UUUUACUCAUUUCAAGGAG 441 2721AACUAGAAGGCUUCUCUCC 152 2721 AACUAGAAGGCUUCUCUCC 152 2739GGAGAGAAGCCUUCUAGUU 442 2739 CACAGUGUUGUGCCCCUUC 153 2739CACAGUGUUGUGCCCCUUC 153 2757 GAAGGGGCACAACACUGUG 443 2757CACUCAUUUUUUUUUGAGG 154 2757 CACUCAUUUUUUUUUGAGG 154 2775CCUCAAAAAAAAAUGAGUG 444 2775 GAGAAGGGGGUCUCUGUUA 155 2775GAGAAGGGGGUCUCUGUUA 155 2793 UAACAGAGACCCCCUUCUC 445 2793AACAUCUAGCCUAAAGUAU 156 2793 AACAUCUAGCCUAAAGUAU 156 2811AUACUUUAGGCUAGAUGUU 446 2811 UACAACUGCCUGGGGGGCA 157 2811UACAACUGCCUGGGGGGCA 157 2829 UGCCCCCCAGGCAGUUGUA 447 2829AGGGUUAGGAAUCUCUUCA 158 2829 AGGGUUAGGAAUCUCUUCA 158 2847UGAAGAGAUUCCUAACCCU 448 2847 ACUACCCUGAUUCUUGAUU 159 2847ACUACCCUGAUUCUUGAUU 159 2865 AAUCAAGAAUCAGGGUAGU 449 2865UCCUGGCUCUACCCUGUCU 160 2865 UCCUGGCUCUACCCUGUCU 160 2883AGACAGGGUAGAGCCAGGA 450 2883 UGUCCCUUUUCUUUGACCA 161 2883UGUCCCUUUUCUUUGACCA 161 2901 UGGUCAAAGAAAAGGGACA 451 2901AGAUCUUUCUCUUCCCUGA 162 2901 AGAUCUUUCUCUUCCCUGA 162 2919UCAGGGAAGAGAAAGAUCU 452 2919 AACGUUUUCUUCUUUCCCU 163 2919AACGUUUUCUUCUUUCCCU 163 2937 AGGGAAAGAAGAAAACGUU 453 2937UGGACAGGCAGCCUCCUUU 164 2937 UGGACAGGCAGCCUCCUUU 164 2955AAAGGAGGCUGCCUGUCCA 454 2955 UGUGUGUAUUCAGAGGCAG 165 2955UGUGUGUAUUCAGAGGCAG 165 2973 CUGCCUCUGAAUACACACA 455 2973GUGAUGACUUGCUGUCCAG 166 2973 GUGAUGACUUGCUGUCCAG 166 2991CUGGACAGCAAGUCAUCAC 456 2991 GGCAGCUCCCUCCUGCACA 167 2991GGCAGCUCCCUCCUGCACA 167 3009 UGUGCAGGAGGGAGCUGCC 457 3009ACAGAAUGCUCAGGGUCAC 168 3009 ACAGAAUGCUCAGGGUCAC 168 3027GUGACCCUGAGCAUUCUGU 458 3027 CUGAACCACUGCUUCUCUU 169 3027CUGAACCACUGCUUCUCUU 169 3045 AAGAGAAGCAGUGGUUCAG 459 3045UUUGAAAGUAGAGCUAGCU 170 3045 UUUGAAAGUAGAGCUAGCU 170 3063AGCUAGCUCUACUUUCAAA 460 3063 UGCCACUUUCACGUGGCCU 171 3063UGCCACUUUCACGUGGCCU 171 3081 AGGCCACGUGAAAGUGGCA 461 3081UCCGCAGUGUCUCCACCUA 172 3081 UCCGCAGUGUCUCCACCUA 172 3099UAGGUGGAGACACUGCGGA 462 3099 ACACCCCUGUGCUCCCCUG 173 3099ACACCCCUGUGCUCCCCUG 173 3117 CAGGGGAGCACAGGGGUGU 463 3117GCCACACUGAUGGCUCAAG 174 3117 GCCACACUGAUGGCUCAAG 174 3135CUUGAGCCAUCAGUGUGGC 464 3135 GACAAGGCUGGCAAACCCU 175 3135GACAAGGCUGGCAAACCCU 175 3153 AGGGUUUGCCAGCCUUGUC 465 3153UCCCAGAAACAUCUCUGGC 176 3153 UCCCAGAAACAUCUCUGGC 176 3171GCCAGAGAUGUUUCUGGGA 466 3171 CCCAGAAAGCCUCUCUCUC 177 3171CCCAGAAAGCCUCUCUCUC 177 3189 GAGAGAGAGGCUUUCUGGG 467 3189CCCUCCCUCUCUCAUGAGG 178 3189 CCCUCCCUCUCUCAUGAGG 178 3207CCUCAUGAGAGAGGGAGGG 468 3207 GCACAGCCAAGCCAAGCGC 179 3207GCACAGCCAAGCCAAGCGC 179 3225 GCGCUUGGCUUGGCUGUGC 469 3225CUCAUGUUGAGCCAGUGGG 180 3225 CUCAUGUUGAGCCAGUGGG 180 3243CCCACUGGCUCAACAUGAG 470 3243 GCCAGCCACAGAGCAAAAG 181 3243GCCAGCCACAGAGCAAAAG 181 3261 CUUUUGCUCUGUGGCUGGC 471 3261GAGGGUUUAUUUUCAGUCC 182 3261 GAGGGUUUAUUUUCAGUCC 182 3279GGACUGAAAAUAAACCCUC 472 3279 CCCUCUCUCUGGGUCAGAA 183 3279CCCUCUCUCUGGGUCAGAA 183 3297 UUCUGACCCAGAGAGAGGG 473 3297ACCAGAGGGCAUGCUGAAU 184 3297 ACCAGAGGGCAUGCUGAAU 184 3315AUUCAGCAUGCCCUCUGGU 474 3315 UGCCCCCUGCUUACUUGGU 185 3315UGCCCCCUGCUUACUUGGU 185 3333 ACCAAGUAAGCAGGGGGCA 475 3333UGAGGGUGCCCCGCCUGAG 186 3333 UGAGGGUGCCCCGCCUGAG 186 3351CUCAGGCGGGGCACCCUCA 476 3351 GUCAGUGCUCUCAGCUGGC 187 3351GUCAGUGCUCUCAGCUGGC 187 3369 GCCAGCUGAGAGCACUGAC 477 3369CAGUGCAAUGCUUGUAGAA 188 3369 CAGUGCAAUGCUUGUAGAA 188 3387UUCUACAAGCAUUGCACUG 478 3387 AGUAGGAGGAAACAGUUCU 189 3387AGUAGGAGGAAACAGUUCU 189 3405 AGAACUGUUUCCUCCUACU 479 3405UCACUGGGAAGAAGCAAGG 190 3405 UCACUGGGAAGAAGCAAGG 190 3423CCUUGCUUCUUCCCAGUGA 480 3423 GGCAAGAACCCAAGUGCCU 191 3423GGCAAGAACCCAAGUGCCU 191 3441 AGGCACUUGGGUUCUUGCC 481 3441UCACCUCGAAAGGAGGCCC 192 3441 UCACCUCGAAAGGAGGCCC 192 3459GGGCCUCCUUUCGAGGUGA 482 3459 CUGUUCCCUGGAGUCAGGG 193 3459CUGUUCCCUGGAGUCAGGG 193 3477 CCCUGACUCCAGGGAACAG 483 3477GUGAACUGCAAAGCUUUGG 194 3477 GUGAACUGCAAAGCUUUGG 194 3495CCAAAGCUUUGCAGUUCAC 484 3495 GCUGAGACCUGGGAUUUGA 195 3495GCUGAGACCUGGGAUUUGA 195 3513 UCAAAUCCCAGGUCUCAGC 485 3513AGAUACCACAAACCCUGCU 196 3513 AGAUACCACAAACCCUGCU 196 3531AGCAGGGUUUGUGGUAUCU 486 3531 UGAACACAGUGUCUGUUCA 197 3531UGAACACAGUGUCUGUUCA 197 3549 UGAACAGACACUGUGUUCA 487 3549AGCAAACUAACCAGCAUUC 198 3549 AGCAAACUAACCAGCAUUC 198 3567GAAUGCUGGUUAGUUUGCU 488 3567 CCCUACAGCCUAGGGCAGA 199 3567CCCUACAGCCUAGGGCAGA 199 3585 UCUGCCCUAGGCUGUAGGG 489 3585ACAAUAGUAUAGAAGUCUG 200 3585 ACAAUAGUAUAGAAGUCUG 200 3603CAGACUUCUAUACUAUUGU 490 3603 GGAAAAAAACAAAAACAGA 201 3603GGAAAAAAACAAAAACAGA 201 3621 UCUGUUUUUGUUUUUUUCC 491 3621AAUUUGAGAACCUUGGACC 202 3621 AAUUUGAGAACCUUGGACC 202 3639GGUCCAAGGUUCUCAAAUU 492 3639 CACUCCUGUCCCUGUAGCU 203 3639CACUCCUGUCCCUGUAGCU 203 3657 AGCUACAGGGACAGGAGUG 493 3657UCAGUCAUCAAAGCAGAAG 204 3657 UCAGUCAUCAAAGCAGAAG 204 3675CUUCUGCUUUGAUGACUGA 494 3675 GUCUGGCUUUGCUCUAUUA 205 3675GUCUGGCUUUGCUCUAUUA 205 3693 UAAUAGAGCAAAGCCAGAC 495 3693AAGAUUGGAAAUGUACACU 206 3693 AAGAUUGGAAAUGUACACU 206 3711AGUGUACAUUUCCAAUCUU 496 3711 UACCAAACACUCAGUCCAC 207 3711UACCAAACACUCAGUCCAC 207 3729 GUGGACUGAGUGUUUGGUA 497 3729CUGUUGAGCCCCAGUGCUG 208 3729 CUGUUGAGCCCCAGUGCUG 208 3747CAGCACUGGGGCUCAACAG 498 3747 GGAAGGGAGGAAGGCCUUU 209 3747GGAAGGGAGGAAGGCCUUU 209 3765 AAAGGCCUUCCUCCCUUCC 499 3765UCUUCUGUGUUAAUUGCGU 210 3765 UCUUCUGUGUUAAUUGCGU 210 3783ACGCAAUUAACACAGAAGA 500 3783 UAGAGGCUACAGGGGUUAG 211 3783UAGAGGCUACAGGGGUUAG 211 3801 CUAACCCCUGUAGCCUCUA 501 3801GCCUGGACUAAAGGCAUCC 212 3801 GCCUGGACUAAAGGCAUCC 212 3819GGAUGCCUUUAGUCCAGGC 502 3819 CUUGUCUUUUGAGCUAUUC 213 3819CUUGUCUUUUGAGCUAUUC 213 3837 GAAUAGCUCAAAAGACAAG 503 3837CACCUCAGUAGAAAAGGAU 214 3837 CACCUCAGUAGAAAAGGAU 214 3855AUCCUUUUCUACUGAGGUG 504 3855 UCUAAGGGAAGAUCACUGU 215 3855UCUAAGGGAAGAUCACUGU 215 3873 ACAGUGAUCUUCCCUUAGA 505 3873UAGUUUAGUUCUGUUGACC 216 3873 UAGUUUAGUUCUGUUGACC 216 3891GGUCAACAGAACUAAACUA 506 3891 CUGUGCACCUACCCCUUGG 217 3891CUGUGCACCUACCCCUUGG 217 3909 CCAAGGGGUAGGUGCACAG 507 3909GAAAUGUCUGCUGGUAUUU 218 3909 GAAAUGUCUGCUGGUAUUU 218 3927AAAUACCAGCAGACAUUUC 508 3927 UCUAAUUCCACAGGUCAUC 219 3927UCUAAUUCCACAGGUCAUC 219 3945 GAUGACCUGUGGAAUUAGA 509 3945CAGAUGCCUGCUUGAUAAU 220 3945 CAGAUGCCUGCUUGAUAAU 220 3963AUUAUCAAGCAGGCAUCUG 510 3963 UAUAUAAACAAUAAAAACA 221 3963UAUAUAAACAAUAAAAACA 221 3981 UGUUUUUAUUGUUUAUAUA 511 3981AACUUUCACUUCUUCCUAU 222 3981 AACUUUCACUUCUUCCUAU 222 3999AUAGGAAGAAGUGAAAGUU 512 3999 UUGUAAUCGUGUGCCAUGG 223 3999UUGUAAUCGUGUGCCAUGG 223 4017 CCAUGGCACACGAUUACAA 513 4017GAUCUGAUCUGUACCAUGA 224 4017 GAUCUGAUCUGUACCAUGA 224 4035UCAUGGUACAGAUCAGAUC 514 4035 ACCCUACAUAAGGCUGGAU 225 4035ACCCUACAUAAGGCUGGAU 225 4053 AUCCAGCCUUAUGUAGGGU 515 4053UGGCACCUCAGGCUGAGGG 226 4053 UGGCACCUCAGGCUGAGGG 226 4071CCCUCAGCCUGAGGUGCCA 516 4071 GCCCCAAUGUAUGUGUGGC 227 4071GCCCCAAUGUAUGUGUGGC 227 4089 GCCACACAUACAUUGGGGC 517 4089CUGUGGGUGUGGGUGGGAG 228 4089 CUGUGGGUGUGGGUGGGAG 228 4107CUCCCACCCACACCCACAG 518 4107 GUGUGUCUGCUGAGUAAGG 229 4107GUGUGUCUGCUGAGUAAGG 229 4125 CCUUACUCAGCAGACACAC 519 4125GAACACGAUUUUCAAGAUU 230 4125 GAACACGAUUUUCAAGAUU 230 4143AAUCUUGAAAAUCGUGUUC 520 4143 UCUAAAGCUCAAUUCAAGU 231 4143UCUAAAGCUCAAUUCAAGU 231 4161 ACUUGAAUUGAGCUUUAGA 521 4161UGACACAUUAAUGAUAAAC 232 4161 UGACACAUUAAUGAUAAAC 232 4179GUUUAUCAUUAAUGUGUCA 522 4179 CUCAGAUCUGAUCAAGAGU 233 4179CUCAGAUCUGAUCAAGAGU 233 4197 ACUCUUGAUCAGAUCUGAG 523 4197UCCGGAUUUCUAACAGUCC 234 4197 UCCGGAUUUCUAACAGUCC 234 4215GGACUGUUAGAAAUCCGGA 524 4215 CCUGCUUUGGGGGGUGUGC 235 4215CCUGCUUUGGGGGGUGUGC 235 4233 GCACACCCCCCAAAGCAGG 525 4233CUGACAACUUAGCUCAGGU 236 4233 CUGACAACUUAGCUCAGGU 236 4251ACCUGAGCUAAGUUGUCAG 526 4251 UGCCUUACAUCUUUUCUAA 237 4251UGCCUUACAUCUUUUCUAA 237 4269 UUAGAAAAGAUGUAAGGCA 527 4269AUCACAGUGUUGCAUAUGA 238 4269 AUCACAGUGUUGCAUAUGA 238 4287UCAUAUGCAACACUGUGAU 528 4287 AGCCUGCCCUCACUCCCUC 239 4287AGCCUGCCCUCACUCCCUC 239 4305 GAGGGAGUGAGGGCAGGCU 529 4305CUGCAGAAUCCCUUUGCAC 240 4305 CUGCAGAAUCCCUUUGCAC 240 4323GUGCAAAGGGAUUCUGCAG 530 4323 CCUGAGACCCUACUGAAGU 241 4323CCUGAGACCCUACUGAAGU 241 4341 ACUUCAGUAGGGUCUCAGG 531 4341UGGCUGGUAGAAAAAGGGG 242 4341 UGGCUGGUAGAAAAAGGGG 242 4359CCCCUUUUUCUACCAGCCA 532 4359 GCCUGAGUGGAGGAUUAUC 243 4359GCCUGAGUGGAGGAUUAUC 243 4377 GAUAAUCCUCCACUCAGGC 533 4377CAGUAUCACGAUUUGCAGG 244 4377 CAGUAUCACGAUUUGCAGG 244 4395CCUGCAAAUCGUGAUACUG 534 4395 GAUUCCCUUCUGGGCUUCA 245 4395GAUUCCCUUCUGGGCUUCA 245 4413 UGAAGCCCAGAAGGGAAUC 535 4413AUUCUGGAAACUUUUGUUA 246 4413 AUUCUGGAAACUUUUGUUA 246 4431UAACAAAAGUUUCCAGAAU 536 4431 AGGGCUGCUUUUCUUAAGU 247 4431AGGGCUGCUUUUCUUAAGU 247 4449 ACUUAAGAAAAGCAGCCCU 537 4449UGCCCACAUUUGAUGGAGG 248 4449 UGCCCACAUUUGAUGGAGG 248 4467CCUCCAUCAAAUGUGGGCA 538 4467 GGUGGAAAUAAUUUGAAUG 249 4467GGUGGAAAUAAUUUGAAUG 249 4485 CAUUCAAAUUAUUUCCACC 539 4485GUAUUUGAUUUAUAAGUUU 250 4485 GUAUUUGAUUUAUAAGUUU 250 4503AAACUUAUAAAUCAAAUAC 540 4503 UUUUUUUUUUUUUGGGUUA 251 4503UUUUUUUUUUUUUGGGUUA 251 4521 UAACCCAAAAAAAAAAAAA 541 4521AAAAGAUGGUUGUAGCAUU 252 4521 AAAAGAUGGUUGUAGCAUU 252 4539AAUGCUACAACCAUCUUUU 542 4539 UUAAAAUGGAAAAUUUUCU 253 4539UUAAAAUGGAAAAUUUUCU 253 4557 AGAAAAUUUUCCAUUUUAA 543 4557UCCUUGGUUUGCUAGUAUC 254 4557 UCCUUGGUUUGCUAGUAUC 254 4575GAUACUAGCAAACCAAGGA 544 4575 CUUGGGUGUAUUCUCUGUA 255 4575CUUGGGUGUAUUCUCUGUA 255 4593 UACAGAGAAUACACCCAAG 545 4593AAGUGUAGCUCAAAUAGGU 256 4593 AAGUGUAGCUCAAAUAGGU 256 4611ACCUAUUUGAGCUACACUU 546 4611 UCAUCAUGAAAGGUUAAAA 257 4611UCAUCAUGAAAGGUUAAAA 257 4629 UUUUAACCUUUCAUGAUGA 547 4629AAAGCGAGGUGGCCAUGUU 258 4629 AAAGCGAGGUGGCCAUGUU 258 4647AACAUGGCCACCUCGCUUU 548 4647 UAUGCUGGUGGUUAAGGCC 259 4647UAUGCUGGUGGUUAAGGCC 259 4665 GGCCUUAACCACCAGCAUA 549 4665CAGGGCCUCUCCAACCACU 260 4665 CAGGGCCUCUCCAACCACU 260 4683AGUGGUUGGAGAGGCCCUG 550 4683 UGUGCCACUGACUUGCUGU 261 4683UGUGCCACUGACUUGCUGU 261 4701 ACAGCAAGUCAGUGGCACA 551 4701UGUGACCCUGGGCAAGUCA 262 4701 UGUGACCCUGGGCAAGUCA 262 4719UGACUUGCCCAGGGUCACA 552 4719 ACUUAACUAUAAGGUGCCU 263 4719ACUUAACUAUAAGGUGCCU 263 4737 AGGCACCUUAUAGUUAAGU 553 4737UCAGUUUUCCUUCUGUUAA 264 4737 UCAGUUUUCCUUCUGUUAA 264 4755UUAACAGAAGGAAAACUGA 554 4755 AAAUGGGGAUAAUAAUACU 265 4755AAAUGGGGAUAAUAAUACU 265 4773 AGUAUUAUUAUCCCCAUUU 555 4773UGACCUACCUCAAAGGGCA 266 4773 UGACCUACCUCAAAGGGCA 266 4791UGCCCUUUGAGGUAGGUCA 556 4791 AGUUUUGAGGCAUGACUAA 267 4791AGUUUUGAGGCAUGACUAA 267 4809 UUAGUCAUGCCUCAAAACU 557 4809AUGCUUUUUAGAAAGCAUU 268 4809 AUGCUUUUUAGAAAGCAUU 268 4827AAUGCUUUCUAAAAAGCAU 558 4827 UUUGGGAUCCUUCAGCACA 269 4827UUUGGGAUCCUUCAGCACA 269 4845 UGUGCUGAAGGAUCCCAAA 559 4845AGGAAUUCUCAAGACCUGA 270 4845 AGGAAUUCUCAAGACCUGA 270 4863UCAGGUCUUGAGAAUUCCU 560 4863 AGUAUUUUUUAUAAUAGGA 271 4863AGUAUUUUUUAUAAUAGGA 271 4881 UCCUAUUAUAAAAAAUACU 561 4881AAUGUCCACCAUGAACUUG 272 4881 AAUGUCCACCAUGAACUUG 272 4899CAAGUUCAUGGUGGACAUU 562 4899 GAUACGUCCGUGUGUCCCA 273 4899GAUACGUCCGUGUGUCCCA 273 4917 UGGGACACACGGACGUAUC 563 4917AGAUGCUGUCAUUAGUCUA 274 4917 AGAUGCUGUCAUUAGUCUA 274 4935UAGACUAAUGACAGCAUCU 564 4935 AUAUGGUUCUCCAAGAAAC 275 4935AUAUGGUUCUCCAAGAAAC 275 4953 GUUUCUUGGAGAACCAUAU 565 4953CUGAAUGAAUCCAUUGGAG 276 4953 CUGAAUGAAUCCAUUGGAG 276 4971CUCCAAUGGAUUCAUUCAG 566 4971 GAAGCGGUGGAUAACUAGC 277 4971GAAGCGGUGGAUAACUAGC 277 4989 GCUAGUUAUCCACCGCUUC 567 4989CCAGACAAAAUUUGAGAAU 278 4989 CCAGACAAAAUUUGAGAAU 278 5007AUUCUCAAAUUUUGUCUGG 568 5007 UACAUAAACAACGCAUUGC 279 5007UACAUAAACAACGCAUUGC 279 5025 GCAAUGCGUUGUUUAUGUA 569 5025CCACGGAAACAUACAGAGG 280 5025 CCACGGAAACAUACAGAGG 280 5043CCUCUGUAUGUUUCCGUGG 570 5043 GAUGCCUUUUCUGUGAUUG 281 5043GAUGCCUUUUCUGUGAUUG 281 5061 CAAUCACAGAAAAGGCAUC 571 5061GGGUGGGAUUUUUUCCCUU 282 5061 GGGUGGGAUUUUUUCCCUU 282 5079AAGGGAAAAAAUCCCACCC 572 5079 UUUUAUGUGGGAUAUAGUA 283 5079UUUUAUGUGGGAUAUAGUA 283 5097 UACUAUAUCCCACAUAAAA 573 5097AGUUACUUGUGACAAAAAU 284 5097 AGUUACUUGUGACAAAAAU 284 5115AUUUUUGUCACAAGUAACU 574 5115 UAAUUUUGGAAUAAUUUCU 285 5115UAAUUUUGGAAUAAUUUCU 285 5133 AGAAAUUAUUCCAAAAUUA 575 5133UAUUAAUAUCAACUCUGAA 286 5133 UAUUAAUAUCAACUCUGAA 286 5151UUCAGAGUUGAUAUUAAUA 576 5151 AGCUAAUUGUACUAAUCUG 287 5151AGCUAAUUGUACUAAUCUG 287 5169 CAGAUUAGUACAAUUAGCU 577 5169GAGAUUGUGUUUGUUCAUA 288 5169 GAGAUUGUGUUUGUUCAUA 288 5187UAUGAACAAACACAAUCUC 578 5187 AAUAAAAGUGAAGUGAAUC 289 5187AAUAAAAGUGAAGUGAAUC 289 5205 GAUUCACUUCACUUUUAUU 579 5201GAAUCUAAAAAAAAAAAAA 290 5201 GAAUCUAAAAAAAAAAAAA 290 5219UUUUUUUUUUUUUAGAUUC 580The 3′-ends of the Upper sequence and the Lower sequence of the siNAconstruct can include an overhang sequence, for example about 1, 2, 3,or 4 nucleotides in length, preferably 2 nucleotides in length, whereinthe overhanging sequence of the lower sequence is optionallycomplementary to a portion of the target sequence. The upper sequence isalso referred to as the sense strand, whereas the lower sequence is alsoreferred to as the antisense strand. The upper and lower sequences inthe#Table can further comprise a chemical modification having FormulaeI-VII, such as exemplary siNA constructs shown in Figures 4 and 5, orhaving modifications described in Table IV or any combination thereof.

TABLE III SCD Synthetic Modified siNA Constructs Target Seq Seq PosTarget ID Cmpd# Aliases Sequence ID 993 GAUAUGCUGUGGUGCUUAAUGCC 58131021 SCD:995U21 sense siNA UAUGCUGUGGUGCUUAAUGTT 597 2518ACUGCUGGACAUGAGAUGGAGAG 582 31022 SCD:2520U21 sense siNAUGCUGGACAUGAGAUGGAGTT 598 3783 UAGAGGCUACAGGGGUUAGCCUG 583 31023SCD:3785U21 sense siNA GAGGCUACAGGGGUUAGCCTT 599 4772CUGACCUACCUCAAAGGGCAGUU 584 31024 SCD:4774U21 sense siNAGACCUACCUCAAAGGGCAGTT 600 660 ACACAAUGGCAUUCCAGAAUGAU 585 31635SCD:662U21 sense siNA ACAAUGGCAUUCCAGAAUGTT 601 2879UGUCUGUCCCUUUUCUUUGACCA 586 31636 SCD:2881U21 sense siNAUCUGUCCCUUUUCUUUGACTT 602 3557 AACCAGCAUUCCCUACAGCCUAG 587 31637SCD:3559U21 sense siNA CCAGCAUUCCCUACAGCCUTT 603 4970AGAAGCGGUGGAUAACUAGCCAG 588 31638 SCD:4972U21 sense siNAAAGCGGUGGAUAACUAGCCTT 604 993 GAUAUGCUGUGGUGCUUAAUGCC 581 31097SCD:1013L21 antisense siNA CAUUAAGCACCACAGCAUATT 605 (995C) 2518ACUGCUGGACAUGAGAUGGAGAG 582 31098 SCD:2538L21 antisense siNACUCCAUCUCAUGUCCAGCATT 606 (2520C) 3783 UAGAGGCUACAGGGGUUAGCCUG 583 31099SCD:3803L21 antisense siNA GGCUAACCCCUGUAGCCUCTT 607 (3785C) 4772CUGACCUACCUCAAAGGGCAGUU 584 31100 SCD:4792L21 antisense siNACUGCCCUUUGAGGUAGGUCTT 608 (4774C) 660 ACACAAUGGCAUUCCAGAAUGAU 585 31639SCD:680L21 antisense siNA CAUUCUGGAAUGCCAUUGUTT 609 (662C) 2879UGUCUGUCCCUUUUCUUUGACCA 586 31640 SCD:2899L21 antisense siNAGUCAAAGAAAAGGGACAGATT 610 (2881C) 3557 AACCAGCAUUCCCUACAGCCUAG 587 31641SCD:3577L21 antisense siNA AGGCUGUAGGGAAUGCUGGTT 611 (3559C) 4970AGAAGCGGUGGAUAACUAGCCAG 588 31642 SCD:4990L21 antisense siNAGGCUAGUUAUCCACCGCUUTT 612 (4972C) 993 GAUAUGCUGUGGUGCUUAAUGCC 581 30873SCD:995U21 sense siNA B uAuGcuGuGGuGcuuAAuGTT B 613 stab04 2518ACUGCUGGACAUGAGAUGGAGAG 582 30874 SCD:2520U21 sense siNA BuGcuGGAcAuGAGAuGGAGTT B 614 stab04 3783 UAGAGGCUACAGGGGUUAGCCUG 58330875 SCD:3785U21 sense siNA B GAGGcuAcAGGGGuuAGccTT B 615 stab04 4772CUGACCUACCUCAAAGGGCAGUU 584 30876 SCD:4774U21 sense siNA BGAccuAccucAAAGGGcAGTT B 616 stab04 660 ACACAAUGGCAUUCCAGAAUGAU 585SCD:662U21 sense siNA B AcAAuGGcAuuccAGAAuGTT B 617 stab04 2879UGUCUGUCCCUUUUCUUUGACCA 586 SCD:2881U21 sense siNA BucuGucccuuuucuuuGAcTT B 618 stab04 3557 AACCAGCAUUCCCUACAGCCUAG 587SCD:3559U21 sense siNA stab04 B ccAGcAuucccuAcAGccuTT B 619 4970AGAAGCGGUGGAUAACUAGCCAG 588 SCD:4972U21 sense siNA stab04 BAAGcGGuGGAuAAcuAGccTT B 620 993 GAUAUGCUGUGGUGCUUAAUGCC 581 30877SCD:1013L21 antisense siNA cAuuAAGcAccAcAGcAuATsT 621 (995C) stab05 2518ACUGCUGGACAUGAGAUGGAGAG 582 30878 SCD:2538L21 antisense siNAcuccAucucAuGuccAGcATsT 622 (2520C) stab05 3783 UAGAGGCUACAGGGGUUAGCCUG583 30879 SCD:3803L21 antisense siNA GGcuAAccccuGuAGccucTsT 623 (3785C)stab05 4772 CUGACCUACCUCAAAGGGCAGUU 584 30880 SCD:4792L21 antisense siNAcuGcccuuuGAGGuAGGucTsT 624 (4774C) stab05 660 ACACAAUGGCAUUCCAGAAUGAU585 SCD:680L21 antisense siNA cAuucuGGAAuGccAuuGuTsT 625 (662C) stab052879 UGUCUGUCCCUUUUCUUUGACCA 586 SCD:2899L21 antisense siNAGucAAAGAAAAGGGAcAGATsT 626 (2881C) stab05 3557 AACCAGCAUUCCCUACAGCCUAG587 SCD:3577L21 antisense siNA AGGcuGuAGGGAAuGcuGGTsT 627 (3559C) stab054970 AGAAGCGGUGGAUAACUAGCCAG 588 SCD:4990L21 antisense siNAGGcuAGuuAuccAccGcuuTsT 628 (4972C) stab05 993 GAUAUGCUGUGGUGCUUAAUGCC581 SCD:995U21 sense siNA stab07 B uAuGcuGuGGuGcuuAAuGTT B 629 2518ACUGCUGGACAUGAGAUGGAGAG 582 SCD:2520U21 sense siNA stab07 BuGcuGGAcAuGAGAuGGAGTT B 630 3783 UAGAGGCUACAGGGGUUAGCCUG 583 SCD:3785U21sense siNA stab07 B GAGGcuAcAGGGGuuAGccTT B 631 4772CUGACCUACCUCAAAGGGCAGUU 584 SCD:4774U21 sense siNA stab07 BGAccuAccucAAAGGGcAGTT B 632 660 ACACAAUGGCAUUCCAGAAUGAU 585 SCD:662U21sense siNA stab07 B AcAAuGGcAuuccAGAAuGTT B 633 2879UGUCUGUCCCUUUUCUUUGACCA 586 SCD:2881U21 sense siNA stab07 BucuGucccuuuucuuuGAcTT B 634 3557 AACCAGCAUUCCCUACAGCCUAG 587 31871SCD:3559U21 sense siNA stab07 B ccAGcAuucccuAcAGccuTT B 635 4970AGAAGCGGUGGAUAACUAGCCAG 588 SCD:4972U21 sense siNA stab07 BAAGcGGuGGAuAAcuAGccTT B 636 993 GAUAUGCUGUGGUGCUUAAUGCC 581 SCD:1013L21antisense siNA cAuuAAGcAccAcAGcAuATsT 637 (995C) stab11 2518ACUGCUGGACAUGAGAUGGAGAG 582 SCD:2538L21 antisense siNAcuccAucucAuGuccAGcATsT 638 (2520C) stab11 3783 UAGAGGCUACAGGGGUUAGCCUG583 SCD:3803L21 antisense siNA GGcuAAccccuGuAGccucTsT 639 (3785C) stab114772 CUGACCUACCUCAAAGGGCAGUU 584 SCD:4792L21 antisense siNAcuGcccuuuGAGGuAGGucTsT 640 (4774C) stab11 660 ACACAAUGGCAUUCCAGAAUGAU585 SCD:680L21 antisense siNA cAuucuGGAAuGccAuuGuTsT 641 (662C) stab112879 UGUCUGUCCCUUUUCUUUGACCA 586 SCD:2899L21 antisense siNAGucAAAGAAAAGGGAcAGATsT 642 (2881C) stab11 3557 AACCAGCAUUCCCUACAGCCUAG587 SCD:3577L21 antisense siNA AGGcuGuAGGGAAuGcuGGTsT 643 (3559C) stab114970 AGAAGCGGUGGAUAACUAGCCAG 588 SCD:4990L21 antisense siNAGGcuAGuuAuccAccGcuuTsT 644 (4972C) stab11 993 GAUAUGCUGUGGUGCUUAAUGCC581 SCD:995U21 sense siNA stab18 B uAuGcuGuGGuGcuuAAuGTT B 645 2518ACUGCUGGACAUGAGAUGGAGAG 582 SCD:2520U21 sense siNA stab18 BuGcuGGAcAuGAGAuGGAGTT B 646 3783 UAGAGGCUACAGGGGUUAGCCUG 583 SCD:3785U21sense siNA stab18 B GAGGcuAcAGGGGuuAGccTT B 647 4772CUGACCUACCUCAAAGGGCAGUU 584 SCD:4774U21 sense siNA stab18 BGAccuAccucAAAGGGcAGTT B 648 660 ACACAAUGGCAUUCCAGAAUGAU 585 SCD:662U21sense siNA stab18 B AcAAuGGcAuuccAGAAuGTT B 649 2879UGUCUGUCCCUUUUCUUUGACCA 586 SCD:2881U21 sense siNA stab18 BucuGucccuuuucuuuGAcTT B 650 3557 AACCAGCAUUCCCUACAGCCUAG 587 SCD:3559U21sense siNA stab18 B ccAGcAuucccuAcAGccuTT B 651 4970AGAAGCGGUGGAUAACUAGCCAG 588 SCD:4972U21 sense siNA stab18 BAAGcGGuGGAuAAcuAGccTT B 652 993 GAUAUGCUGUGGUGCUUAAUGCC 581 SCD:1013L21antisense siNA cAuuAAGcAccAcAGcAuATsT 653 (995C) stab08 2518ACUGCUGGACAUGAGAUGGAGAG 582 SCD:2538L21 antisense siNAcuccAucucAuGuccAGcATsT 654 (2520C) stab08 3783 UAGAGGCUACAGGGGUUAGCCUG583 SCD:3803L21 antisense siNA GGcuAAccccuGuAGccucTsT 655 (3785C) stab084772 CUGACCUACCUCAAAGGGCAGUU 584 SCD:4792L21 antisense siNAcuGcccuuuGAGGuAGGucTsT 656 (4774C) stab08 660 ACACAAUGGCAUUCCAGAAUGAU585 SCD:680L21 antisense siNA cAuucuGGAAuGccAuuGuTsT 657 (662C) stab082879 UGUCUGUCCCUUUUCUUUGACCA 586 SCD:2899L21 antisense siNAGucAAAGAAAAGGGAcAGATsT 658 (2881C) stab08 3557 AACCAGCAUUCCCUACAGCCUAG587 31877 SCD:3577L21 antisense siNA AGGcuGuAGGGAAuGcuGGTsT 659 (3559C)stab08 4970 AGAAGCGGUGGAUAACUAGCCAG 588 SCD:4990L21 antisense siNAGGcuAGuuAuccAccGcuuTsT 660 (4972C) stab08 993 GAUAUGCUGUGGUGCUUAAUGCC581 SCD:995U21 sense siNA stab09 B UAUGCUGUGGUGCUUAAUGTT B 661 2518ACUGCUGGACAUGAGAUGGAGAG 582 SCD:2520U21 sense siNA stab09 BUGCUGGACAUGAGAUGGAGTT B 662 3783 UAGAGGCUACAGGGGUUAGCCUG 583 SCD:3785U21sense siNA stab09 B GAGGCUACAGGGGUUAGCCTT B 663 4772CUGACCUACCUCAAAGGGCAGUU 584 SCD:4774U21 sense siNA stab09 BGACCUACCUCAAAGGGCAGTT B 664 660 ACACAAUGGCAUUCCAGAAUGAU 585 SCD:662U21sense siNA stab09 B ACAAUGGCAUUCCAGAAUGTT B 665 2879UGUCUGUCCCUUUUCUUUGACCA 586 SCD:2881U21 sense siNA stab09 BUCUGUCCCUUUUCUUUGACTT B 666 3557 AACCAGCAUUCCCUACAGCCUAG 587 SCD:3559U21sense siNA stab09 B CCAGCAUUCCCUACAGCCUTT B 667 4970AGAAGCGGUGGAUAACUAGCCAG 588 SCD:4972U21 sense siNA stab09 BAAGCGGUGGAUAACUAGCCTT B 668 993 GAUAUGCUGUGGUGCUUAAUGCC 581 SCD:1013L21antisense siNA CAUUAAGCACCACAGCAUATsT 669 (995C) stab10 2518ACUGCUGGACAUGAGAUGGAGAG 582 SCD:2538L21 antisense siNACUCCAUCUCAUGUCCAGCATsT 670 (2520C) stab10 3783 UAGAGGCUACAGGGGUUAGCCUG583 SCD:3803L21 antisense siNA GGCUAACCCCUGUAGCCUCTsT 671 (3785C) stab104772 CUGACCUACCUCAAAGGGCAGUU 584 SCD:4792L21 antisense siNACUGCCCUUUGAGGUAGGUCTsT 672 (4774C) stab10 660 ACACAAUGGCAUUCCAGAAUGAU585 SCD:680L21 antisense siNA CAUUCUGGAAUGCCAUUGUTsT 673 (662C) stab102879 UGUCUGUCCCUUUUCUUUGACCA 586 SCD:2899L21 antisense siNAGUCAAAGAAAAGGGACAGATsT 674 (2881C) stab10 3557 AACCAGCAUUCCCUACAGCCUAG587 SCD:3577L21 antisense siNA AGGCUGUAGGGAAUGCUGGTsT 675 (3559C) stab104970 AGAAGCGGUGGAUAACUAGCCAG 588 SCD:4990L21 antisense siNAGGCUAGUUAUCCACCGCUUTsT 676 (4972C) stab10 993 GAUAUGCUGUGGUGCUUAAUGCC581 SCD:1013L21 antisense siNA cAuuAAGcAccAcAGcAuATT B 677 (995C) stab192518 ACUGCUGGACAUGAGAUGGAGAG 582 SCD:2538L21 antisense siNAcuccAucucAuGuccAGcATT B 678 (2520C) stab19 3783 UAGAGGCUACAGGGGUUAGCCUG583 SCD:3803L21 antisense siNA GGcuAAccccuGuAGccucTT B 679 (37850)stab19 4772 CUGACCUACCUCAAAGGGCAGUU 584 SCD:4792L21 antisense siNAcuGcccuuuGAGGuAGGucTT B 680 (4774C) stab19 660 ACACAAUGGCAUUCCAGAAUGAU585 SCD:680L21 antisense siNA cAuucuGGAAuGccAuuGuTT B 681 (662C) stab192879 UGUCUGUCCCUUUUCUUUGACCA 586 SCD:2899L21 antisense siNAGucAAAGAAAAGGGAcAGATT B 682 (2881C) stab19 3557 AACCAGCAUUCCCUACAGCCUAG587 SCD:3577L21 antisense siNA AGGcuGuAGGGAAuGcuGGTT B 683 (3559C)stab19 4970 AGAAGCGGUGGAUAACUAGCCAG 588 SCD:4990L21 antisense siNAGGcuAGuuAuccAccGcuuTT B 684 (4972C) stab19 993 GAUAUGCUGUGGUGCUUAAUGCC581 SCD:1013L21 antisense siNA CAUUAAGCACCACAGCAUATT B 685 (9950) stab222518 ACUGCUGGACAUGAGAUGGAGAG 582 SCD:2538L21 antisense siNACUCCAUCUCAUGUCCAGCATT B 686 (2520C) stab22 3783 UAGAGGCUACAGGGGUUAGCCUG583 SCD:3803L21 antisense siNA GGCUAACCCCUGUAGCCUCTT B 687 (3785C)stab22 4772 CUGACCUACCUCAAAGGGCAGUU 584 SCD:4792L21 antisense siNACUGCCCUUUGAGGUAGGUCU B 688 (4774C) stab22 660 ACACAAUGGCAUUCCAGAAUGAU585 SCD:680L21 antisense siNA CAUUCUGGAAUGCCAUUGUTT B 689 (662C) stab222879 UGUCUGUCCCUUUUCUUUGACCA 586 SCD:2899L21 antisense siNAGUCAAAGAAAAGGGACAGATT B 690 (2881C) stab22 3557 AACCAGCAUUCCCUACAGCCUAG587 SCD:3577L21 antisense siNA AGGCUGUAGGGAAUGCUGGTT B 691 (3559C)stab22 4970 AGAAGCGGUGGAUAACUAGCCAG 588 SCD:4990L21 antisense siNAGGCUAGUUAUCCACCGCUUTT B 692 (4972C) stab22 645 UUCUGAUCAUUGCCAACACAAUG589 SCD:647U21 sense siNA CUGAUCAUUGCCAACACAATT 693 1192CACCACAUUCUUCAUUGAUUGCA 590 SCD:1194U21 sense siNA CCACAUUCUUCAUUGAUUGTT694 2023 GUUUGGCAAUGCUAAUUCAAUGC 591 SCD:2025U21 sense siNAUUGGCAAUGCUAAUUCAAUTT 695 2728 AGGCUUCUCUCCACAGUGUUGUG 592 SCD:2730U21sense siNA GCUUCUCUCCACAGUGUUGTT 696 3554 ACUAACCAGCAUUCCCUACAGCC 593SCD:3556U21 sense siNA UAACCAGCAUUCCCUACAGTT 697 4316CUUUGCACCUGAGACCCUACUGA 594 SCD:4318U21 sense siNA UUGCACCUGAGACCCUACUTT698 4318 UUGCACCUGAGACCCUACUGAAG 595 SCD:4320U21 sense siNAGCACCUGAGACCCUACUGATT 699 4775 ACCUACCUCAAAGGGCAGUUUUG 596 SCD:4777U21sense siNA CUACCUCAAAGGGCAGUUUTT 700 645 UUCUGAUCAUUGCCAACACAAUG 589SCD:665L21 antisense siNA UUGUGUUGGCAAUGAUCAGTT 701 (647C) 1192CACCACAUUCUUCAUUGAUUGCA 590 SCD:1212L21 antisense siNACAAUCAAUGAAGAAUGUGGTT 702 (1194C) 2023 GUUUGGCAAUGCUAAUUCAAUGC 591SCD:2043L21 antisense siNA AUUGAAUUAGCAUUGCCAATT 703 (2025C) 2728AGGCUUCUCUCCACAGUGUUGUG 592 SCD:2748L21 antisense siNACAACACUGUGGAGAGAAGCTT 704 (2730C) 3554 ACUAACCAGCAUUCCCUACAGCC 593SCD:3574L21 antisense siNA CUGUAGGGAAUGCUGGUUATT 705 (3556C) 4316CUUUGCACCUGAGACCCUACUGA 594 SCD:4336L21 antisense siNAAGUAGGGUCUCAGGUGCAATT 706 (4318C) 4318 UUGCACCUGAGACCCUACUGAAG 595SCD:4338L21 antisense siNA UCAGUAGGGUCUCAGGUGCTT 707 (4320C) 4775ACCUACCUCAAAGGGCAGUUUUG 596 SCD:4795L21 antisense siNAAAACUGCCCUUUGAGGUAGTT 708 (4777C) 645 UUCUGAUCAUUGCCAACACAAUG 589SCD:647U21 sense siNA stab04 B cuGAucAuuGccAAcAcAATT B 709 1192CACCACAUUCUUCAUUGAUUGCA 590 SCD:1194U21 sense siNA stab04 BccAcAuucuucAuuGAuuGTT B 710 2023 GUUUGGCAAUGCUAAUUCAAUGC 591 SCD:2025U21sense siNA stab04 B uuGGcAAuGcuAAuucAAuTT B 711 2728AGGCUUCUCUCCACAGUGUUGUG 592 SCD:2730U21 sense siNA stab04 BGcuucucuccAcAGuGuuGTT B 712 3554 ACUAACCAGCAUUCCCUACAGCC 593 SCD:3556U21sense siNA stab04 B uAAccAGcAuucccuAcAGTT B 713 4316CUUUGCACCUGAGACCCUACUGA 594 SCD:4318U21 sense siNA stab04 BuuGcAccuGAGAcccuAcuTT B 714 4318 UUGCACCUGAGACCCUACUGAAG 595 SCD:4320U21sense siNA stab04 B GCAccuGAGAcccuAcuGATT B 715 4775ACCUACCUCAAAGGGCAGUUUUG 596 SCD:4777U21 sense siNA stab04 BcuAccucAAAGGGcAGuuuTT B 716 645 UUCUGAUCAUUGCCAACACAAUG 589 SCD:665L21antisense siNA uuGuGuuGGcAAuGAucAGTsT 717 (647C) stab05 1192CACCACAUUCUUCAUUGAUUGCA 590 SCD:1212L21 antisense siNAcAAucAAuGAAGAAuGuGGTsT 718 (1194C) stab05 2023 GUUUGGCAAUGCUAAUUCAAUGC591 SCD:2043L21 antisense siNA AuuGAAuuAGcAuuGccAATsT 719 (2025C) stab052728 AGGCUUCUCUCCACAGUGUUGUG 592 SCD:2748L21 antisense siNAcAAcAcuGuGGAGAGAAGcTsT 720 (2730C) stab05 3554 ACUAACCAGCAUUCCCUACAGCC593 SCD:3574L21 antisense siNA cuGuAGGGAAuGcuGGuuATsT 721 (3556C) stab054316 CUUUGCACCUGAGACCCUACUGA 594 SCD:4336L21 antisense siNAAGuAGGGucucAGGuGcAATsT 722 (4318C) stab05 4318 UUGCACCUGAGACCCUACUGAAG595 SCD:4338L21 antisense siNA ucAGuAGGGucucAGGuGcTsT 723 (4320C) stab054775 ACCUACCUCAAAGGGCAGUUUUG 596 SCD:4795L21 antisense siNAAAAcuGcccuuuGAGGuAGTsT 724 (4777C) stab05 645 UUCUGAUCAUUGCCAACACAAUG589 SCD:647U21 sense siNA stab07 B cuGAucAuuGccAAcAcAATT B 725 1192CACCACAUUCUUCAUUGAUUGCA 590 SCD:1194U21 sense siNA stab07 BccAcAuucuucAuuGAuuGTT B 726 2023 GUUUGGCAAUGCUAAUUCAAUGC 591 SCD:2025U21sense siNA stab07 B uuGGcAAuGcuAAuucAAuTT B 727 2728AGGCUUCUCUCCACAGUGUUGUG 592 SCD:2730U21 sense siNA stab07 BGcuucucuccAcAGuGuuGTT B 728 3554 ACUAACCAGCAUUCCCUACAGCC 593 SCD:3556U21sense siNA stab07 B uAAccAGcAuucccuAcAGTT B 729 4316CUUUGCACCUGAGACCCUACUGA 594 SCD:4318U21 sense siNA stab07 BuuGcAccuGAGAcccuAcuTT B 730 4318 UUGCACCUGAGACCCUACUGAAG 595 SCD:4320U21sense siNA stab07 B GcAccuGAGAcccuAcuGATT B 731 4775ACCUACCUCAAAGGGCAGUUUUG 596 SCD:4777U21 sense siNA stab07 BcuAccucAAAGGGcAGuuuTT B 732 645 UUCUGAUCAUUGCCAACACAAUG 589 SCD:665L21antisense siNA uuGuGuuGGcAAuGAucAGTsT 733 (647C) stab11 1192CACCACAUUCUUCAUUGAUUGCA 590 SCD:1212L21 antisense siNAcAAucAAuGAAGAAuGuGGTsT 734 (1194C) stab11 2023 GUUUGGCAAUGCUAAUUCAAUGC591 SCD:2043L21 antisense siNA AuuGAAuuAGcAuuGccAATsT 735 (2025C) stab112728 AGGCUUCUCUCCACAGUGUUGUG 592 SCD:2748L21 antisense siNAcAAcAcuGuGGAGAGAAGcTsT 736 (2730C) stab11 3554 ACUAACCAGCAUUCCCUACAGCC593 SCD:3574L21 antisense siNA cuGuAGGGAAuGcuGGuuATsT 737 (3556C) stab114316 CUUUGCACCUGAGACCCUACUGA 594 SCD:4336L21 antisense siNAAGuAGGGucucAGGuGcAATsT 738 (4318C) stab11 4318 UUGCACCUGAGACCCUACUGAAG595 SCD:4338L21 antisense siNA ucAGuAGGGucucAGGuGcTsT 739 (4320C) stab114775 ACCUACCUCAAAGGGCAGUUUUG 596 SCD:4795L21 antisense siNAAAAcuGcccuuuGAGGuAGTsT 740 (4777C) stab11 645 UUCUGAUCAUUGCCAACACAAUG589 SCD:647U21 sense siNA B cuGAucAuuGccAAcAcAATT B 741 stab18 1192CACCACAUUCUUCAUUGAUUGCA 590 SCD:1194U21 sense siNA stab18 BccAcAuucuucAuuGAuuGTT B 742 2023 GUUUGGCAAUGCUAAUUCAAUGC 591 SCD:2025U21sense siNA stab18 B uuGGcAAuGcuAAuucAAuTT B 743 2728AGGCUUCUCUCCACAGUGUUGUG 592 SCD:2730U21 sense siNA stab18 BGcuucucuccAcAGuGuuGTT B 744 3554 ACUAACCAGCAUUCCCUACAGCC 593 SCD:3556U21sense siNA stab18 B uAAccAGcAuucccuAcAGTT B 745 4316CUUUGCACCUGAGACCCUACUGA 594 SCD:4318U21 sense siNA stab18 BuuGcAccuGAGAcccuAcuTT B 746 4318 UUGCACCUGAGACCCUACUGAAG 595 SCD:4320U21sense siNA stab18 B GcAccuGAGAcccuAcuGATT B 747 4775ACCUACCUCAAAGGGCAGUUUUG 596 SCD:4777U21 sense siNA stab18 BcuAccucAAAGGGcAGuuuTT B 748 645 UUCUGAUCAUUGCCAACACAAUG 589 SCD:665L21antisense siNA uuGuGuuGGcAAuGAucAGTsT 749 (647C) stab08 1192CACCACAUUCUUCAUUGAUUGCA 590 SCD:1212L21 antisense siNAcAAucAAuGAAGAAuGuGGTsT 750 (1194C) stab08 2023 GUUUGGCAAUGCUAAUUCAAUGC591 SCD:2043L21 antisense siNA AuuGAAuuAGcAuuGccAATsT 751 (2025C) stab082728 AGGCUUCUCUCCACAGUGUUGUG 592 SCD:2748L21 antisense siNAcAAcAcuGuGGAGAGAAGcTsT 752 (2730C) stab08 3554 ACUAACCAGCAUUCCCUACAGCC593 SCD:3574L21 antisense siNA cuGuAGGGAAuGcuGGuuATsT 753 (3556C) stab084316 CUUUGCACCUGAGACCCUACUGA 594 SCD:4336L21 antisense siNAAGuAGGGucucAGGuGcAATsT 754 (4318C) stab08 4318 UUGCACCUGAGACCCUACUGAAG595 SCD:4338L21 antisense siNA ucAGuAGGGucucAGGuGcTsT 755 (4320C) stab084775 ACCUACCUCAAAGGGCAGUUUUG 596 SCD:4795L21 antisense siNAAAAcuGcccuuuGAGGuAGTsT 756 (4777C) stab08 645 UUCUGAUCAUUGCCAACACAAUG589 37108 SCD:647U21 sense siNA stab09 B CUGAUCAUUGCCAACACAATT B 7571192 CACCACAUUCUUCAUUGAUUGCA 590 37109 SCD:1194U21 sense siNA stab09 BCCACAUUCUUCAUUGAUUGTT B 758 2023 GUUUGGCAAUGCUAAUUCAAUGC 591 37110SCD:2025U21 sense siNA stab09 B UUGGCAAUGCUAAUUCAAUTT B 759 2728AGGCUUCUCUCCACAGUGUUGUG 592 37111 SCD:2730U21 sense siNA stab09 BGCUUCUCUCCACAGUGUUGTT B 760 3554 ACUAACCAGCAUUCCCUACAGCC 593 37112SCD:3556U21 sense siNA stab09 B UAACCAGCAUUCCCUACAGTT B 761 4316CUUUGCACCUGAGACCCUACUGA 594 37113 SCD:4318U21 sense siNA stab09 BUUGCACCUGAGACCCUACUTT B 762 4318 UUGCACCUGAGACCCUACUGAAG 595 37114SCO:4320U21 sense siNA stab09 B GCACCUGAGACCCUACUGATT B 763 4775ACCUACCUCAAAGGGCAGUUUUG 596 37115 SCD:4777U21 sense siNA stab09 BCUACCUCAAAGGGCAGUUUTT B 764 645 UUCUGAUCAUUGCCAACACAAUG 589 SCD:665L21antisense siNA UUGUGUUGGCAAUGAUCAGTsT 765 (647C) stab10 1192CACCACAUUCUU0AUUGAUUGCA 590 SCD:1212L21 antisense siNACAAUCAAUGAAGAAUGUGGTsT 766 (1194C) stab10 2023 GUUUGGCAAUGCUAAUUCAAUGC591 SCD:2043L21 antisense siNA AUUGAAUUAGOAUUGOCAATsT 767 (2025C) stab102728 AGGCUUCUCUCCACAGUGUUGUG 592 SCD:2748L21 antisense siNACAACACUGUGGAGAGAAGCTsT 768 (2730C) stab10 3554 ACUAACCAGCAUUCCCUACAGCC593 SCD:3574L21 antisense siNA CUGUAGGGAAUGCUGGUUATsT 769 (3556C) stab104316 CUUUGCACCUGAGACCCUACUGA 594 SCD:4336L21 antisense siNAAGUAGGGUCUCAGGUGCAATsT 770 (4318C) stab10 4318 UUGCACCUGAGACCCUACUGAAG595 SCD:4338L21 antisense siNA UCAGUAGGGUCUCAGGUGCTsT 771 (4320C) stab104775 ACCUACCUCAAAGGGCAGUUUUG 596 SCD:4795L21 antisense siNAAAACUGCCCUUUGAGGUAGTsT 772 (4777C) stab10 645 UUCUGAUCAUUGCCAACACAAUG589 SCD:665L21 antisense siNA uuGuGuuGGcAAuGAucAGTT B 773 (647C) stab191192 CACCACAUUCUUCAUUGAUUGCA 590 SCD:1212L21 antisense siNAcAAucAAuGAAGAAuGuGGTT B 774 (1194C) stab19 2023 GUUUGGCAAUGCUAAUUCAAUGC591 SCD:2043L21 antisense siNA AuuGAAuuAGcAuuGccAATT B 775 (2025C)stab19 2728 AGGCUUCUCUCCACAGUGUUGUG 592 SCD:2748L21 antisense siNAcAAcAcuGuGGAGAGAAGcTT B 776 (2730C) stab19 3554 ACUAACCAGCAUUCCCUACAGCC593 SCD:3574L21 antisense siNA cuGuAGGGAAuGcuGGuuATT B 777 (3556C)stab19 4316 CUUUGCACCUGAGACCCUACUGA 594 SCD:4336L21 antisense siNAAGuAGGGucucAGGuGcAATT B 778 (4318C) stab19 4318 UUGCACCUGAGACCCUACUGAAG595 SCD:4338L21 antisense siNA ucAGuAGGGucucAGGuGcTT B 779 (4320C)stab19 4775 ACCUACCUCAAAGGGCAGUUUUG 596 SCD:4795L21 antisense siNAAAAcuGcccuuuGAGGuAGTT B 780 (4777C) stab19 645 UUCUGAUCAUUGCCAACACAAUG589 37116 SCD:665L21 antisense siNA UUGUGUUGGCAAUGAUCAGTT B 781 (647C)stab22 1192 CACCACAUUCUUCAUUGAUUGCA 590 37117 SCD:1212L21 antisense siNACAAUCAAUGAAGAAUGUGGTT B 782 (1194C) stab22 2023 GUUUGGCAAUGCUAAUUCAAUGC591 37118 SCD:2043L21 antisense siNA AUUGAAUUAGCAUUGCCAATT B 783 (2025C)2728 AGGCUUCUCUCCACAGUGUUGUG 592 37119 SCD:2748L21 antisense siNACAACACUGUGGAGAGAAGCTT B 784 (2730C) stab22 3554 ACUAACCAGCAUUCCCUACAGCC593 37120 SCD:3574L21 antisense siNA CUGUAGGGAAUGCUGGUUATT B 785 (3556C)stab22 4316 CUUUGCACCUGAGACCCUACUGA 594 37121 SCD:4336L21 antisense siNAAGUAGGGUCUCAGGUGCAATT B 786 (4318C) stab22 4318 UUGCACCUGAGACCCUACUGAAG595 37122 SCD:4338L21 antisense siNA UCAGUAGGGUCUCAGGUGCTT B 787 (4320C)stab22 4775 ACCUACCUCAAAGGGCAGUUUUG 596 37123 SCD:4795L21 antisense siNAAAACUGCCCUUUGAGGUAGTT B 788 (4777C) stab22Uppercase = ribonucleotideu,c = 2′-deoxy-2′-fluoro U,CT = thymidineB = inverted deoxy abasics = phosphorothioate linkageA = deoxy AdenosineG = deoxy GuanosineG = 2′-O-methyl GuanosineA = 2′-O-methyl Adenosine

TABLE IV Non-limiting examples of Stabilization Chemistries forchemically modified siNA constructs pyri- Chemistry midine Purine cap p= S Strand “Stab 00” Ribo Ribo TT at 3′- S/AS ends “Stab 1” Ribo Ribo —5 at 5′-end S/AS 1 at 3′-end “Stab 2” Ribo Ribo — All Usually ASlinkages “Stab 3” 2′-fluoro Ribo — 4 at 5′-end Usually S 4 at 3′-end“Stab 4” 2′-fluoro Ribo 5′ and 3′- — Usually S ends “Stab 5” 2′-fluoroRibo — 1 at 3′-end Usually AS “Stab 6” 2′-O- Ribo 5′ and 3′- — Usually SMethyl ends “Stab 7” 2′-fluoro 2′-deoxy 5′ and 3′- — Usually S ends“Stab 8” 2′-fluoro 2′-O- — 1 at 3′-end S/AS Methyl “Stab 9” Ribo Ribo 5′and 3′- — Usually S ends “Stab 10” Ribo Ribo — 1 at 3′-end Usually AS“Stab 11” 2′-fluoro 2′-deoxy — 1 at 3′-end Usually AS “Stab 12”2′-fluoro LNA 5′ and 3′- Usually S ends “Stab 13” 2′-fluoro LNA 1 at3′-end Usually AS “Stab 14” 2′-fluoro 2′-deoxy 2 at 5′-end Usually AS 1at 3′-end “Stab 15” 2′-deoxy 2′-deoxy 2 at 5′-end Usually AS 1 at 3′-end“Stab 16” Ribo 2′-O- 5′ and 3′- Usually S Methyl ends “Stab 17” 2′-O-2′-O- 5′ and 3′- Usually S Methyl Methyl ends “Stab 18” 2′-fluoro 2′-O-5′ and 3′- Usually S Methyl ends “Stab 19” 2′-fluoro 2′-O- 3′-end S/ASMethyl “Stab 20” 2′-fluoro 2′-deoxy 3′-end Usually AS “Stab 21”2′-fluoro Ribo 3′-end Usually AS “Stab 22” Ribo Ribo 3′-end Usually AS“Stab 23” 2′-fluoro* 2′-deoxy* 5′ and 3′- Usually S ends “Stab 24”2′-fluoro* 2′-O- — 1 at 3′-end S/AS Methyl* “Stab 25” 2′-fluoro* 2′-O- —1 at 3′-end S/AS Methyl* “Stab 26” 2′-fluoro* 2′-O- — S/AS Methyl* “Stab27” 2′-fluoro* 2′-O- 3′-end S/AS Methyl* “Stab 28” 2′-fluoro* 2′-O-3′-end S/AS Methyl* “Stab 29” 2′-fluoro* 2′-O- 1 at 3′-end S/AS Methyl*“Stab 30” 2′-fluoro* 2′-O- S/AS Methyl* “Stab 31” 2′-fluoro* 2′-O-3′-end S/AS Methyl* “Stab 32” 2′-fluoro 2′-O- S/AS MethylCAP = any terminal cap, see for example FIG. 10.All Stab 00-32 chemistries can comprise 3′-terminal thymidine (TT)residuesAll Stab 00-32 chemistries typically comprise about 21 nucleotides, butcan vary as described herein.S = sense strandAS = antisense strand*Stab 23 has a single ribonucleotide adjacent to 3′-CAP*Stab 24 and Stab 28 have a single ribonucleotide at 5′-terminus*Stab 25, Stab 26, and Stab 27 have three ribonucleotides at 5′-terminus*Stab 29, Stab 30, and Stab 31, any purine at first three nucleotidepositions from 5′-terminus are ribonucleotidesp = phosphorothioate linkage

TABLE V Reagent Equivalents Amount Wait Time* DNA Wait Time* 2′-O-methylWait Time*RNA A. 2.5 μmol Synthesis Cycle ABI 394 InstrumentPhosphoramidites 6.5 163 μL 45 sec 2.5 min 7.5 min S-Ethyl Tetrazole23.8 238 μL 45 sec 2.5 min 7.5 min Acetic Anhydride 100 233 μL 5 sec 5sec 5 sec N-Methyl 186 233 μL 5 sec 5 sec 5 sec Imidazole TCA 176 2.3 mL21 sec 21 sec 21 sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage12.9 645 μL 100 sec 300 sec 300 sec Acetonitrile NA 6.67 mL NA NA NA B.0.2 μmol Synthesis Cycle ABI 394 Instrument Phosphoramidites 15 31 μL 45sec 233 sec 465 sec S-Ethyl Tetrazole 38.7 31 μL 45 sec 233 min 465 secAcetic Anhydride 655 124 μL 5 sec 5 sec 5 sec N-Methyl 1245 124 μL 5 sec5 sec 5 sec Imidazole TCA 700 732 μL 10 sec 10 sec 10 sec Iodine 20.6244 μL 15 sec 15 sec 15 sec Beaucage 7.7 232 μL 100 sec 300 sec 300 secAcetonitrile NA 2.64 mL NA NA NA C. 0.2 μmol Synthesis Cycle 96 wellInstrument Amount: Wait Time* Equivalents: DNA/ DNA/2′-O- Wait Time*2′-O- Wait Time* Reagent 2′-O-methyl/Ribo methyl/Ribo DNA methyl RiboPhosphoramidites 22/33/66 40/60/120 μL 60 sec 180 sec 360 sec S-EthylTetrazole 70/105/210 40/60/120 μL 60 sec 180 min 360 sec AceticAnhydride 265/265/265 50/50/50 μL 10 sec 10 sec 10 sec N-Methyl502/502/502 50/50/50 μL 10 sec 10 sec 10 sec Imidazole TCA 238/475/475250/500/500 μL 15 sec 15 sec 15 sec Iodine 6.8/6.8/6.8 80/80/80 μL 30sec 30 sec 30 sec Beaucage 34/51/51 80/120/120 100 sec 200 sec 200 secAcetonitrile NA 1150/1150/1150 μL NA NA NA*Wait time does not include contact time during delivery.*Tandem synthesis utilizes double coupling of linker molecule

1. A chemically synthesized double stranded short interfering nucleicacid (siNA) molecule that directs cleavage of a SCD RNA via RNAinterference (RNAi), wherein: a) each strand of said siNA molecule isabout 18 to about 23 nucleotides in length; and b) one strand of saidsiNA molecule comprises nucleotide sequence having sufficientcomplementarity to said SCD RNA for the siNA molecule to direct cleavageof the SCD RNA via RNA interference.
 2. The siNA molecule of claim 1,wherein said siNA molecule comprises no ribonucleotides.
 3. The siNAmolecule of claim 1, wherein said siNA molecule comprises one or moreribonucleotides.
 4. The siNA molecule of claim 1, wherein one strand ofsaid double-stranded siNA molecule comprises a nucleotide sequence thatis complementary to a nucleotide sequence of a SCD gene or a portionthereof, and wherein a second strand of said double-stranded siNAmolecule comprises a nucleotide sequence substantially similar to thenucleotide sequence or a portion thereof of said SCD RNA.
 5. The siNAmolecule of claim 4, wherein each strand of the siNA molecule comprisesabout 18 to about 23 nucleotides, and wherein each strand comprises atleast about 19 nucleotides that are complementary to the nucleotides ofthe other strand.
 6. The siNA molecule of claim 1, wherein said siNAmolecule comprises an antisense region comprising a nucleotide sequencethat is complementary to a nucleotide sequence of a SCD gene or aportion thereof, and wherein said siNA further comprises a sense region,wherein said sense region comprises a nucleotide sequence substantiallysimilar to the nucleotide sequence of said SCD gene or a portionthereof.
 7. The siNA molecule of claim 6, wherein said antisense regionand said sense region comprise about 18 to about 23 nucleotides, andwherein said antisense region comprises at least about 18 nucleotidesthat are complementary to nucleotides of the sense region.
 8. The siNAmolecule of claim 1, wherein said siNA molecule comprises a sense regionand an antisense region, and wherein said antisense region comprises anucleotide sequence that is complementary to a nucleotide sequence ofRNA encoded by a SCD gene, or a portion thereof, and said sense regioncomprises a nucleotide sequence that is complementary to said antisenseregion.
 9. The siNA molecule of claim 6, wherein said siNA molecule isassembled from two separate oligonucleotide fragments wherein onefragment comprises the sense region and a second fragment comprises theantisense region of said siNA molecule.
 10. The siNA molecule of claim6, wherein said sense region is connected to the antisense region via alinker molecule.
 11. The siNA molecule of claim 10, wherein said linkermolecule is a polynucleotide linker.
 12. The siNA molecule of claim 10,wherein said linker molecule is a non-nucleotide linker.
 13. The siNAmolecule of claim 6, wherein pyrimidine nucleotides in the sense regionare 2′-O-methyl pyrimidine nucleotides.
 14. The siNA molecule of claim6, wherein purine nucleotides in the sense region are 2′-deoxy purinenucleotides.
 15. The siNA molecule of claim 6, wherein pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides.
 16. The siNA molecule of claim 9, wherein thefragment comprising said sense region includes a terminal cap moiety ata 5′-end, a 3′-end, or both of the 5′ and 3′ ends of the fragmentcomprising said sense region.
 17. The siNA molecule of claim 16, whereinsaid terminal cap moiety is an inverted deoxy abasic moiety.
 18. ThesiNA molecule of claim 6, wherein pyrimidine nucleotides of saidantisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides.
 19. ThesiNA molecule of claim 6, wherein purine nucleotides of said antisenseregion are 2′-O-methyl purine nucleotides.
 20. The siNA molecule ofclaim 6, wherein purine nucleotides present in said antisense regioncomprise 2′-deoxy- purine nucleotides.
 21. The siNA molecule of claim18, wherein said antisense region comprises a phosphorothioateinternucleotide linkage at the 3′ end of said antisense region.
 22. ThesiNA molecule of claim 6, wherein said antisense region comprises aglyceryl modification at a 3′ end of said antisense region.
 23. The siNAmolecule of claim 9, wherein each of the two fragments of said siNAmolecule comprise about 21 nucleotides.
 24. The siNA molecule of claim23, wherein about 19 nucleotides of each fragment of the siNA moleculeare base-paired to the complementary nucleotides of the other fragmentof the siNA molecule and wherein at least two 3′ terminal nucleotides ofeach fragment of the siNA molecule are not base-paired to thenucleotides of the other fragment of the siNA molecule.
 25. The siNAmolecule of claim 24, wherein each of the two 3′ terminal nucleotides ofeach fragment of the siNA molecule are 2′-deoxy-pyrimidines.
 26. ThesiNA molecule of claim 25, wherein said 2′-deoxy-pyrimidine is2′-deoxy-thymidine.
 27. The siNA molecule of claim 23, wherein all ofthe about 21 nucleotides of each fragment of the siNA molecule arebase-paired to the complementary nucleotides of the other fragment ofthe siNA molecule.
 28. The siNA molecule of claim 23, wherein about 19nucleotides of the antisense region are base-paired to the nucleotidesequence of the RNA encoded by a SCD gene or a portion thereof.
 29. ThesiNA molecule of claim 23, wherein about 21 nucleotides of the antisenseregion are base-paired to the nucleotide sequence of the RNA encoded bya SCD gene or a portion thereof.
 30. The siNA molecule of claim 9,wherein a 5′-end of the fragment comprising said antisense regionoptionally includes a phosphate group.
 31. A composition comprising thesiNA molecule of claim 1 in an pharmaceutically acceptable carrier ordiluent.
 32. A siNA according to claim 1 wherein the SCD RNA comprisesGenbank Accession No. NM_(—)005063.2.
 33. A siNA according to claim 1wherein said siNA comprises any of SEQ ID NOs. 1-806.
 34. A compositioncomprising the siNA of claim 32 together with a pharmaceuticallyacceptable carrier or diluent.
 35. A composition comprising the siNA ofclaim 33 together with a pharmaceutically acceptable carrier or diluent.